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生醫奈米影像技術
Biomedical Molecular Imaging
Cheng-Chung Chang
生醫奈米影像技術 課程內容(共6小時)
¾
光與能量(Spectra)
¾螢光原理與應用
¾螢光顯微鏡與共軛聚焦螢光顯微鏡(Confocal)
¾螢光共振能量轉換 (FRET)
¾冷光共振能量轉換 (BRET)
¾分子級螢光感測劑 (Biosensor)
¾螢光有機奈米粒(FON)
生醫奈米影像技術 實驗內容(共6小時)
(實驗一):螢光電泳 共3小時
(實驗二): 細胞組織,染色體螢光影像 共3小時
Molecular Imaging Program at Stanford (MIPS) (http://mips.stanford.edu)
分子造影（Molecular Imaging）
定義：在未受損的活體，以分子與分子以下的層級，用影像進行生物反應過程型態表
現、特性及量化的觀察。產生的影像反映細胞及分子路徑，以及疾病在真實生理
環境內表現的體內（in vivo）機轉，是一個多重領域的新知識。
特點：體內動物模式更能夠了解生物過程中的耐受性（Tolerances）、互補性
（Complementation）與重複性等各種現象。也就是分子影像更容易觀察某一特
定基因的功能與作用，且由於是在完整的活體中進行，所以是以更實際的方式，
在複雜、動態的生物系統中進行。
藥物發展:分子造影也可以發揮極大作用。在動物模式進行化合物的毒理測試及有效
性測試時，藉由將潛在藥物進行標幟造影，使得通常在表現型（phenotypic）轉
成顯而易見前，就已可得到清楚、有意義的研究結果，加快進入人體試驗的速
度，這也使臨床前試驗加快排除生物分布（Biodistribution）不良或是藥物動
力學（Pharmacokinetics）不佳的藥物。
最終的目標:是以一個單一的裝置，產生最後融合解剖與生理資訊的三維影像，但最
大的挑戰是在如何克服從一個30公克的小動物造影，延伸到70公斤人體的應用。
分類：
¾ 核醫學: γ射線
正子發射斷層掃描(Positron Emission Tomography, PET)
單光子發射斷層掃描(Single-Photon Emission Computed Tomography,
SPECT)。
¾ 磁振造影（Magnetic Resonance Imaging, MRI）:質子自旋
¾ 電腦斷層掃描（Computed Tomography Imaging, CT）: X光(射線)
¾ 超音波分子影像學: 超音波微泡造影劑
¾
光學造影: 螢光顯微鏡（Fluorescence Microscopy）
化學冷光儀（Luminometry）
非侵入式Non-intrusive的體內（in vivo）造影，藉由光子取得活體的生物訊息。
非侵入式活體分子影像系統 (Non Invasion In Vivo Imaging System)
生物冷光與螢光影像兩者最大的差異就是在螢光影像由於組織自體螢光
（Autofluorescence），使觀察到的背景訊號較生物冷光更為顯著。
光學分子造影進展策略:
‡標的生物螢光探針（targeted bioluminescence probes）
‡近紅外光螢光物質（near-infrared fluorochromes）
‡可活化近紅外光螢光物質（activatable near-infrared fluorochromes）
‡red-shifted螢光蛋白（red-shifted fluorescent proteins）
光學技術最顯著的優勢:
‡多樣(不同光譜)特性探針，進行多通道造影
‡低成本動物模式報導基因（reporter gene）表現研究的選擇
生物冷光（Bioluminescence）與螢光造影（Fluorescence Imaging）:
‡螢光造影主要優點:在活體及固定細胞或組織，不需任何基質就可進行觀察，可做
為一個報告者（reporter）。但是螢光造影系統不具量化能力，且影像資訊有表面
加權的特性，也就是愈接近表面會較結構深的亮度亮，都造成應用上的限制。光學
‡生物冷光造影的主要優點:由於光發射不會被背景影響，所以能夠偵測低層訊號，
可快速並簡單的進行造影。易於操作，影像擷取時間短（通常10-60秒），非常適合
進行大量造影試驗的進行。
影像技術
使用電磁波形式
PET
高能γ射線
SPECT
低能γ射線
Optical
Bio-luminescence Imaging
可見光
Optical
Fluorescence Imaging
可見光或近紅外線
MRI
無線電波
CT
X射線
Ultrasound
高頻聲波
Genes & Development；工研院IEK生醫組
電磁波?!
生醫奈米影像技術
Biomedical Molecular Imaging
I. 光與能量 (Light and Energy)
Cheng-Chung Chang
自然界-光
Luminescence
‧ Theroluminescence (熱發光)(incandescence,熾熱光,電燈泡 3000K,
gas excitation 氣體激發)
‧ Triboluminescence (摩擦發光)機械發光(Mechano—luminescence)
‧ Electroluminescence (電激發光)-LED, OLED
‧ Photoluminescence-- (光激發而發光)
-(fluorescence (on/off), phosphorescence (stay))
‧ Chemiluminescence (化學發光) -light stick
‧ Bioluminescence (生物發光)一fire fly
Forms of Energy
1.
2.
3.
4.
5.
6.
7.
Mechanical( Kinetic and potential)機械能(動能和位能)
Chemical 化學能
Heat
熱能
Electric 電能
Light
光能
Sound 聲能
Nuclear 核能
People have learned how to change energy from one form to
another so that we can do work more easily and live more
comfortably.
Properties
Electric and magnetic field
Electromagnetic wave
Periodic motion
Direction (3x108 m/sec)
Properties
• Particle : Photoelectric Effect
• Wave : Diffraction Pattern
‹
It can be thought of as both a particle and a wave, so called
“particle-wave duality”
‹
Lower energy (longer wavelength) light acts predominately like a wave
‹
High energy (shorter wavelength) light acts predominately like a particle
Application
Source
Materials
detector
能量
質量
E=MC2
光速
(3x108 m/sec )2
= 9x1016 (m/sec)2
愛因斯坦——相對論——質能方程式
Definitions and Range
Energy of photon
E = hν=hc/λ
（λ:wavelength)
Production of X-rays
•
•
•
•
X-rays are produced when high-speed electrons
are suddenly slowed down
Can be caused by the electron striking a metal
target
Emitted only by high energy processes
Can pass through lower density materials
Light (solar)
White Light is made up of
different colours
何謂遠紅外線？
美國醫學物理學家協會謝楠柱表示，遠紅外線是太陽光線中不可視的電磁波，它的
波長是8-14μm。根據最近美國太空總署對遠紅外線的研究發現，遠紅外線對人體具
有良好的治療作用，因為它可以滲透入皮膚下4-5cm深度，引起細胞共振
（Resonance & Absorption）而產生溫熱效應。
細胞共振
細胞受到遠紅外線之感應發生共振，使身體皮下之溫度增高，造成微絲血管擴張，
增進血液循環，加速新陳代謝，令有害物質排出體外，使破損細胞迅速回復正常，
達到治療的效果。
SONY-TR250PK (DV)和KAYA 37毫米PF4濾光镜
紅外線攝影的基本原則其實跟一般攝影是一樣的，都是利用光線照在物體上反射經
過鏡片到相機內成像，區別只是在可見光與紅外光的不同罷了。
數位紅外線攝影
傳統的紅外線攝影要靠紅外線專用底片，而數位相機用的 CCD 或 CMOS 本身就可感
應到紅外線 ，只是在一般的狀況下由於可見光的光量遠大於紅外光所以看不出紅外
線效應( 另一個原因是許多 DC 都裝有 IR cut filter )。
紅外線濾鏡( Infrared Filter ) -在鏡頭前加裝紅外線濾鏡後，底片或 CCD 便只
看到紅外光 ，這就是簡單的紅外線攝影原理 。
當然並不是所有衣服都能被透視，當數純棉料最安全而不能被紅外線功能透視，尼龍
及混棉織料都可以達到透視效果，特別是在游泳池等地的濕衣服更加清楚，純尼龍及
絲質衣服的透視度更是高達75%以上。
為什麼拍出來的紅外線相片跟大家在電視看到的醫療、軍事紅外線影像不同呢？
紅外線影像在醫療、軍事方面，主要用來作透視、目標追蹤、熱量深測等，應用
的是生物、汽車因本身發熱而產生的遠紅外線。數碼相機只能夠感應景物反射陽
光中的近紅外線，因此物件本身的熱量不會對相片有太大影響。同時在晚上由於
沒有陽光照射，景物沒有反射近紅外線，所以拍攝紅外線夜景是不可能的。
沒用的偽裝。
但是我們偉大的國軍，沒事就拿鐵樂士把車輛
噴的漂漂亮亮應付裝檢，原來有防紅外線偵測
的迷彩漆直接報銷，國軍這種怪事多的很。
微波爐
乾燥
高頻是 ”high-frequency”( 縮成成 HF) 。
特高頻”very-high” (VHF)
超高頻”ultra-high” (UHF)
無線電信號以直線的形式前進，可以達到距離約為三十哩
至六十哩。此種無線電波雖然在前進過程中會穿過一些建
築物在跳動中繞過障礙，但最佳接收只在接收天線與發射
天線成一直線時，才能出現。
警察與救護車都使用微波無線電話，和
控制中心保持連絡。很多大公司亦然
900/1800/1900 MHz 三頻
美國 (四頻機)
850 Mz
GSM 900 / PHS 1900 MHz
FM英文全名為
Frequenncy Modulation（調頻)
AM amplitude modulation 調幅
生醫奈米影像技術
Biomedical Molecular Imaging
II. 螢光原理與應用
Cheng-Chung Chang
Principle of Fluorescence:
1.
2.
3.
Energy is absorbed by the atom which becomes excited.
The electron jumps to a higher energy level.
Soon, the electron drops back to the ground state, emitting a
photon (or a packet of light)
- the atom is fluorescing
Energy of photon
E = hν=hc/λ（λ:wavelength），
1
2
3
The probability of a transition
The Franck-Condon energy diagram
the Franck-Condon Principle.
Probabilities maximally overlap in
both the ground and excited states.
‡ Mirror Image Rule.
‡ Absorption spectrum containing multiple peaks.
‡ Quickly undergo relaxation to the lowest vibrational energy level
of the excited state
the naturally
occurring
antimalarial agent
(and first known
fluorophore)
Sir John Fredrick
William Hershel in
1845
In many cases, excitation by high energy photons leads to the population of
higher electronic and vibrational levels (S(2), S(3), etc.), which quickly lose
excess energy as the fluorophore relaxes to the lowest vibrational level of the
first excited state. Hence, emission spectra are generally independent of the
excitation wavelength (some fluorophores emit from higher energy states, but
such activity is rare).
The shorter wavelength ultraviolet absorption peak (310 nanometers) is due to an
excitation transition to the second excited state (from S(0) to S(2)) that quickly
relaxes to the lowest excited state (S(1)). As a consequence, fluorescence
emission occurs exclusively from the lowest excited singlet state (S(1)), resulting
in a spectrum that mirrors the ground to first excited state transition (350
nanometer peak) in quinine and not the entire absorption spectrum.
Because the energy associated with fluorescence emission transitions is typically
less than that of absorption, the resulting emitted photons have less energy and are
shifted to longer wavelengths. This phenomenon is generally known as Stokes
Shift and occurs for virtually all fluorophores commonly employed in solution
investigations.
In practice, the Stokes shift is measured as the difference between the maximum
wavelengths in the excitation and emission spectra of a particular fluorochrome or
fluorophore. The size of the shift varies with molecular structure, but can range from
just a few nanometers to over several hundred nanometers.
For example,
Fluorescein: 20 nm
Quinine: 110 nm
Porphyrins: 200 nanometers.
The existence of Stokes shift is critical to the extremely high sensitivity of
fluorescence imaging measurements. The red emission shift enables the use of
precision bandwidth optical filters to effectively block excitation light from reaching the
detector so the relatively faint fluorescence signal (having a low number of emitted
photons) can be observed against a low-noise background.
Solvent effects
The same organic compound dissolved in solvents of different
polarity. This causes different stabilization of the charged
states, changing the color.
Absorption
Emission
Solvatochromism
European Journal of Organic Chemistry
Volume 2008, Issue 15, Pages 2531-2538
Concentration Effects
The luminescence of perylene bisimide 1a
in toluene is concentration dependent. The
concentrations from left to right are: 10-6,
10-5, 10-4, 10-3 and 10-2 M.
Extinction Coefficient (ε), Quantum Yield (Φ), and Fluorescence Lifetime (τ)
The extinction coefficient is determined by measuring the absorbance at a reference
wavelength (characteristic of the absorbing molecule) for a one molar (M)
concentration (one mole per liter) of the target chemical in a cuvette having a onecentimeter path length.
The quantum yield represents the probability that a given excited fluorochrome will
produce an emitted photon (fluorescence). Quantum yields typically range between
a value of zero and one
Formally, the fluorescence lifetime is defined as the time in which the initial
fluorescence intensity of a fluorophore decays to 1/e (approximately 37 percent) of
the initial intensity
I(t) = Io x e (-t/τ)
In a uniform solvent, fluorescence decay is usually a monoexponential function, as
illustrated by the plots of fluorescence intensity as a function of time in Figures (a)
and (b). More complex systems, such as viable tissues and living cells, contain a
mixed set of environments that often yield multiexponential values (Figure (c))
F = photons emitted/photons absorbed = kf/(kf + knf) = tf/to
Quenching and Photobleaching
A common example of quenching is observed with the collision of an
excited state fluorophore and another (non-fluorescent) molecule in
solution, resulting in deactivation of the fluorophore and return to the
ground state. A wide variety of simple elements and compounds behave
as collisional quenching agents, including oxygen, halogens, amines,
and many electron-deficient organic molecules. The mechanisms for
collisional quenching include electron transfer, spin-orbit coupling, and
intersystem crossing to the excited triplet state.
In contrast to quenching, photobleaching (also termed fading) occurs when a
fluorophore permanently loses the ability to fluoresce due to photon-induced
chemical damage and covalent modification. Upon transition from an excited singlet
state to the excited triplet state, fluorophores may interact with another molecule to
produce irreversible covalent modifications. The triplet state is relatively long-lived
with respect to the singlet state, thus allowing excited molecules a much longer
timeframe to undergo chemical reactions with components in the environment. The
average number of excitation and emission cycles that occur for a particular
fluorophore before photobleaching is dependent upon the molecular structure and
the local environment. Some fluorophores bleach quickly after emitting only a few
photons, while others that are more robust can undergo thousands or millions of
cycles before bleaching.
Bovine pulmonary artery epithelial cells: The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue fluorescence), while
the mitochondria and actin cytoskeleton were stained with MitoTracker Red (red fluorescence) and a phalloidin derivative (green
fluorescence), respectively. Time points were taken in two-minute intervals using a fluorescence filter combination with bandwidths
tuned to excite the three fluorophores simultaneously while also recording the combined emission signals. Note that all three
fluorophores have a relatively high intensity in Figure (a), but the DAPI (blue) intensity starts to drop rapidly at two minutes and is
almost completely gone at six minutes. The mitochondrial and actin stains are more resistant to photobleaching, but the intensity of
both drops over the course of the timed sequence (10 minutes).
生醫奈米影像技術
Biomedical Molecular Imaging
III.螢光顯微鏡與共軛聚焦螢光顯微鏡
Fluorescence Microscopy
Basic Concepts in Fluorescence
British scientist Sir George G. Stokes who first described fluorescence in 1852
Fluorescence is the property of some atoms and molecules to absorb light at a
particular wavelength and to subsequently emit light of longer wavelength after a brief
interval, termed the fluorescence lifetime.
The process of phosphorescence occurs in a manner similar to fluorescence, but with
a much longer excited state lifetime.
Cheng-Chung Chang
Elements
Source
Materials
detector
filter
In a conventional reflected fluorescence light microscope, the light
is usually from mercury arc lamp which provides a mixture of wave
length from UV to red. A emission filter is used to separate desired
wavelength.
The first fluorescence microscopes were developed between 1911 and 1913 by
German physicists Otto Heimstädt and Heinrich Lehmann. These microscopes were
employed to observe autofluorescence in bacteria, animal, and plant tissues.
Stanislav Von Provazek used fluorescence microscopy to study dye binding in fixed
tissues and living cells.
1940s, Albert Coons developed a technique for labeling antibodies with fluorescent
dyes, thus giving birth to the field of immunofluorescence.
By the turn of the twenty-first century, the field of fluorescence microscopy was
responsible for a revolution in cell biology, coupling the power of live cell imaging to
highly specific multiple labeling of individual organelles and macromolecular
complexes with synthetic and genetically encoded fluorescent probes.
Epi-Fluorescence Microscopy
CONFOCAL
共軛焦(confocal)ㄧ辭，是形容光源在接物鏡所形成的焦點，和光電倍增管
( photomultiplier tube ) 所偵測到的ㄧ小光點，是來自相同的物鏡焦點，也就是
照明點與探測點在光學成像上共軛。利用通過光學針孔光圈(pinhole)蒐集來自樣品
聚焦面的光，將非同一聚焦面的光排除於光學針孔光圈外，所形成的影像我們簡單的
稱為共軛焦點影像。
共軛焦顯微鏡所看到的影像只是一個極小的亮點，而不是如傳統顯微鏡所看到的二維
空間影像；利用共軛焦顯微鏡內建的掃描器，可將影像由很多點組成線，很多線組成
面，取得由點厚度所組成的單ㄧ平面影像稱為光切片(optical sections)。光切片配
合電腦輔助運算處理，可呈現出兼具高解析度和低背景雜訊的二維空間相片；或將多
張同平面不同螢光探針的相片重疊，可研究其相對部位的關係；或由一系列不同平面
的相片，可組合成三維空間的立體影像；再加入時間因子，可創造出動態的4D 影
像。
Widefield fluorescence image of a
16 micron thick section of
fluorescently-labeled mouse
kidney. Copyright, J. Waters, 2004
The same specimen show on the
left, taken with the spinning disk
confocal microscope. Copyright, J.
Waters, 2004
Bovine pulmonary artery endothelial (BPAE) cell - F-actin (red) was labeled with
Texas Red phalloidin and nuclei (blue) were labeled with DAPI. Merged projection
from stacks of confocal and multi-photon images.
HT-1080/MT1 tumor cells (green) migrating through a dense matrix of collagen fibers
(blue) in the dermis of a live mouse. A single optical section collected from the multiphoton microscope.
Multi-photon section through a bone marrow cavity in the skull of a live mouse. The
blood plasma is labeled with FITC-dextran (green). Note the rapid blood flow in the
vessel center in the upper portion of the vessel. Rhodamine 6G stains nuclei and
mitchondria in the hematopoietic cells at that adhere within the vessel (the round
spheres inside the green
dextran-filled vessel) and the
surrounding tissue. The cavity is
enclosed in solid bone, which is
not penetrated by rhodamine 6G
and, hence, appears black.
(image won first price
at the April 2003 BioRad image
competition)
Triple labeled bovine pulmonary
artery endothelial (BPAE) cells.
The red mitochondria (stained
with MitoTracker Red CMXRos)
and the green F-actin (labeled
with BODIPY FL phallacidin) were
imaged in confocal mode using
488 nm and 568 nm excitation
wavelengths respectively. The
blue nuclei (labeled with DAPI)
were imaged in multi-photon
mode using 800 nm excitation.
The three colors were merged
into one plane to form this final
image.
Market Fluorescent Probes and Their Applications
Fluorescent probes used in microscopy can be categorised in
several ways.
1. Probes with direct affinity for a specific cellular
component, for instance mitochondria.
2. Probes directed at a selected cellular component using
specific antibodies.
3. Probes reacting to environmental conditions, like pH or
Ca+2 concentration.
4. Enzyme substrates.
5. Other probes.
Viable bovine pulmonary artery endothelial cells simultaneously stained with
BODIPY FL histamine (lysosomal, histamine), MitoTracker Red CMXRos (M7512)
and Hoechst 33342 (H1399, H3570, H21492). Green-fluorescent BODIPY FL
histamine localized to lysosomes, red-fluorescent MitoTracker Red CMXRos
accumulated in the mitochondria, and the blue-fluorescent Hoechst 33342 dye
stained the nuclei. This multiple-exposure image was acquired with bandpass
filters appropriate for fluorescein, the Texas Red dye and DAPI.
Acridine orange (AO) binds to nucleic acids. AO bound to DNA
yields (at a concentration of 10 ( g/ml) green fluorescence while AO
bound to RNA yields red fluorescence.
l ex\l em (MeOH) = 495nm\522nm.
Є (MeOH) = 63,000.
Plasma membrane of a single cell from a cluster was stained with a
lipohilic dye DiI (red) with green auto-fluorescence.
Iex\ Iem (MeOH) = 550nm\565nm.
ε(MeOH) = 145,000.
Mitochondria in glioma cells stained with TMRE. This dye
enters cell in a form of an ester, which is subsequently
hydrolised and the product, tetramethylrhodamine, is
accumulated in mitochondria due to a high membrane
potential.
lex\lem (MeOH) = 549/574nm
Application and Other probes
Live bovine pulmonary artery endothelial
cells were incubated simultaneously with Cell
Trace calcein red-orange AM and MitoTracker
Green FM. The de-esterified calcein redorange is present throughout the cell, whereas
the green-fluorescent MitoTracker Green FM
dye accumulated in the mitochondria. Nuclei
were stained with blue-fluorescent Hoechst
33342.
Abs 570, Flu 610
480, 530
340,450
551,
576
Images of an NIH 3T3 cell showing colocalization of the CoroNa Red sodium
indicator (top panel; sodium indicater) with the MitoTracker Green FM
mitochondrial marker (bottom panel, M7514).
生醫奈米影像技術
Biomedical Molecular Imaging
IV. 螢光共振能量轉換
Fluorescence Resonance Energy
Transfer (FRET)
Cheng-Chung Chang
Fluorescence Resonance Energy Transfer
‡ Permits the investigation of molecular interactions with an optical microscope well beyond
the theoretical resolution limit of light microscopy. ( 50 years ago (Förster, 1948)
‡ Suitable pair of spectrally distinct fluorescent molecules is sufficiently close (between 1
and 10nm) for a radiationless transfer of energy via a dipole-dipole interaction from the
excited fluorophore (donor) to the second fluorophore (acceptor).
‡ Since transfer efficiency has an inverse 6th power dependency upon distance between the
acceptor and donor this technique is inherently sensitive to small physical changes in
proximity.
‡ The Förster distance, defined as the distance between the donor and acceptor where the
average efficiency of energy transfer is 50% .
‡ Commonly, FRET is employed to visualize protein-protein interactions by targeting the
desired cellular constituents with fluorescent dyes, fluorescent antibodies and/or fluorescent
proteins.
‡ A cellular target is labeled with a donor fluorophore while another is labeled with an
acceptor fluorophore. If the fluorophore labeled proteins are intimately associated (See Fig.1),
excitation of the donor molecule will produce an emission at the acceptor wavelength.
(Periasamy and Day, 1999; Sekar and Periasamy, 2003)
FRET Donor and Acceptor Spectral Profiles
FRET schematic using CFP and YFP fusion proteins.
No Energy Transfer: Excitation of a
donor fluorophore (D) sufficiently
separated from its acceptor fluorophore
(A) will result in donor emission.
FRET-Energy Transfer: Excitation of a donor
fluorophore (D) within 10nm of its acceptor
fluorophore (A) will result in acceptor emission and
quenched donor emission.
Important Criterion for Successful FRET
(Periasamy and Day, 1999)
‡ The donor probe should have a high quantum yield.
‡ The fluorophores chosen should have a significant overlap in the donor
emission and acceptor excitation spectra also known as the overlap integral.
‡ The donor and acceptor must be in the proper orientation with respect to one
another. The factor K2, approximated at 2/3 when random orientation of both
fluorophores occurs, is used to express this orientation.
‡ The donor and acceptor must be less than 10 nm from each other.
‡ Note: Although the proteins of interest may truely interact, a number of factors
may result in the donor and acceptor being physically positioned too far from
each other for energy transfer.
The Acceptor Photobleaching Method
‡ A relatively straightforward approach for determining FRET with
conventional and confocal microscopy.
‡ Bleaching of the acceptor fluorophore will dequench the donor and
result in donor emission (See Fig. 2).
Simplified schematic of how acceptor
photobleaching works.
Quenched Donor (FRET): Excitation of a
donor fluorophore that is (D) within 10nm of
its acceptor fluorophore (A) will result in
acceptor emission and quenched donor
emission.
Acceptor Photobleach: Sufficient excitation
to irreversibly photobleach the acceptor
fluorophore (A) eliminates the potential for
the acceptor to participate in energy transfer.
Dequenched Donor: Excitation of the donor
fluorophore (D) following acceptor
photobleaching (A) will result in dequenched
donor emission.
The structural and functional organization of living cells is highly dependant
upon the numerous protein-protein interactions that occur from within. An
understanding of these interactions and their significance is a major
impetus for modern multidisciplinary biological research (Mendelsohn and
Brent, 1999).
FRET has been found to be suitable for determining inter- or intramolecular distances related to during binding, association, conformational
shifts, diffusion and catalysis within cells (Jares-Erijman and Jovin, 2003).
For example, using of FRET to measure viral protein interactions (MeqVil8)
and calcium channel subunit (α-1C).
Cameleons: Calcium Ion Probes
Cameleons are a new class of indicators for calcium ion concentrations in living cells, which
operate through a conformational change that results in fluorescence resonance energy
transfer (FRET) in the presence of calcium ions.
In 1997, Miyawak 等將BFP(或CFP)加到鈣調蛋白的N端，將GFP(或YFP)連接
到M13的c端(M13是來源於骨骼肌肌球蛋白輕鏈激酶的鈣調蛋白結合肽)，構
成“Cameleons”。鈣離子與鈣調蛋白的結合將導致鈣調蛋白與M13的纏繞，從
而減少供體與受體的距離，提高FRET的效率。
TaqMan
PNAS | February 18, 2003 | vol. 100 | no. 4 | 1569-1573
Patrizia Alberti and Jean-Louis Mergny
Nanomolecular machine
FRET with Antibodies
Cy3-Cy5 FRET using acceptor photobleaching
method. Positive control dual-channel image of a cell
labeled with a rabbit anti-α-1C primary antibody and
1:1 ratio of Cy3 and Cy5 donkey anti-rabbit Fab
secondary antibodies. This image is the final frame of a
repeat bleach experiment. A region of interest drawn in
the bleached area (box labeled 1) demonstrated the drop
in Cy5 signal intensity over time (curve D) with
concomitant increase in dequenched Cy3 signal intensity
(curve A). The unbleached region (box labeled 2)
showed no change in Cy3 (donor) or Cy5 (acceptor)
signal intensity during the same experiment.
Ying Shao and Dr. Mary C. Farach-Carson Department of Biological Sciences, University of Delaware.
FRET with Fluorescent Proteins
ECFP-EYFP FRET using acceptor photobleaching method. Paraformaldehyde fixed
chicken fibroblast cells co-transfected to express a MeqVil8-ECFP and MeqVil8-ECFP illustrate
dimerization of this herpes virus. This image is the final frame of a repeat bleach experiment. A
region of interest selecting a single punctate structure of MeqVil8 was drawn in the bleached area
(box labeled 1) and demonstrated the drop in EYFP signal intensity over time (curve D) with
concomitant increase in dequenched ECFP
signal intensity (curve B). The unbleached region
(box labeled 2) showed no significant change in
ECFP (donor) or EYFP (acceptor) signal intensity
during the same experiment.
Jon Anobile and Dr. Carl Schmidt, Department of
Animal and Food Sciences, University of Delaware.
Fluorescence Photobleaching Investigations
Both fluorescence loss in photobleaching (FLIP and the related methodology of
recovery after photobleaching (FRAP) are techniques for observing the movement of
intracellular materials through photobleaching of fluorescence.
A specific area of a floating fluorescent dye on a cell membrane, an organelle
(endoplasmic reticulum and Golgi apparatus) membrane, or a floating fluorescencelabeled protein on these membranes is bleached, and the loss or recovery of
fluorescence is observed to examine fluidity in the lateral direction. The techniques of
FLIP and FRAP are also used to confirm the continuity of membranes.
Recently, a study was conducted in which cytoskeletal proteins were labeled with
fluorescent dye to confirm how turnover (replacement) of proteins constituting
microtubules and intermediate-size filaments (10-nanometer filaments) occurs
(Vikstrom et al., 1992). The techniques presented in this study can be used to
determine whether the turnover occurs at the end or in the middle of the filaments
Graphical presentation of data collected during a FRAP experiment. A baseline of fluorescence
is collected (1) before the photobleaching occurs (arrow) so that the amount of fluorescence is
reduced significantly (2). Over time, the amount of fluorescence in the photobleached area
increases as unbleached molecules diffuse into this area (3). Later, there is a stabilization of the
amount of fluorescence recovery (4) and a flat line is obtained. The percent recovery uses the
formula: (Y/ X) x 100 = % recovery. In the diagram, the percentage of fluorescence lost due to
photobleaching is X and the amount of fluorescence that returned to the bleached area is Y. In
practice, the percent recovery almost never reaches 100%. The lateral mobility is determined by
the slope of the curve (3). The steeper the curve, the faster the recovery and therefore, the more
mobile the molecules.
生醫奈米影像技術
Biomedical Molecular Imaging
V. Biosensor
(Fluorosensor)
Cheng-Chung Chang
A. Conjugating labeling Biosensor
Aldehyde-, Ketone-, & Carboxylate-Reactive Labels
Amine-Reactive Fluorophores, Biotins, Quantum Dots, & Other Labels
Antibody & Protein Labeling Kits
Biotin Labeling Kits & Reagents
Cell Labeling Kits & Reagents
Other Reactive Probes
Peptide Synthesis Reagents
Phycobiliproteins
Thiol-Reactive Fluorophores, Biotins & Other Labels
Bioconjugate Chem. 1997, 8,751-756
Functionalized Tricarbocyanine Dyes as Near-Infrare
Fluorescent Probes for Biomolecules
isothiocyanate
succinimidyl ester
carbocyanine dyes
‡ These dyes containing isothiocyanate and succinimidyl ester functional groups
are reactive toward primary amines and can be used as fluorescent probes for
biologically pertinent compounds such as amino acids and functionalized
dideoxynucleotides.
‡ The absorption and fluorescence maxima occur in the near-IR region of the
spectrum (770-810 nm). The succinimidyl ester proved to be very sensitive to
hydrolysis and was generated in situ to label amino acids.
‡ The isothiocyanates were less susceptible to hydrolysis and were conjugated
using organic modified [40% (v/v) acetonitrile] buffers to amino acids. A dye
with an alkyl isothiocyanate moiety showed conjugation to amino-functionalized
dideoxynucleotide triphosphates.
1,1-Thiocarbonyldiimidazole
•isothiocyanate
S
N
N
N
+
H2N
OH
N
DMF
C
OH
S
N
N2
•Tyramine
30 min
O
N
O
OH
N2
O
O
C
O
N
C
-
O
OH
DCC
anhydrous DMF
3-(p-Hydroxyphenyl)propionic acid
O
O
OH
Labeling of Amino Acids with
Isothiocyanate
A 10-fold excess of 1 or 2 in DMSO was
dissolved in a 0.1 mM solution of amino
acid in borate buffer [13 mM, pH 9.3,
40% (v/v) acetonitrile; 100 uL of 10:40:50
DMSO/acetonitrile/buffer], and the
solution was shaken in the dark at room
temperature. The reaction was analyzed
by analytical HPLC after 12 h.
Labeling of Amino Acids with
Succinimidyl Derivative
Aliquots of the reaction solution were
added in a 10-fold excess to 1 uM
solutions of tryptophan in
carbonate/bicarbonate buffer (67 mM, pH
9.4). Reactions were followed by
analytical HPLC. The reactions were
determined to be complete within 20 min.
1
1
1
1
1
www.caymaneurope.com
www.invitrogen.com
Lightly fixed human epidermoid carcinoma
cells (A431) stained with biotinylated
epidermal growth factor (EGF) complexed to
Texas Red® streptavidin (E3480). An
identical cell preparation stained in the
presence of a 100-fold excess of unlabeled
EGF (E3476) showed no fluorescent signal.
The cytoskeleton of a fixed and permeabilized bovine pulmonary artery endothelial
cell detected using mouse monoclonal anti–a-tubulin antibody (Cat. no. A11126),
visualized with Alexa Fluor® 647 goat anti–mouse IgG antibody (Cat. no. A21235) and
pseudocolored magenta. Endogenous biotin in the mitochondria was labeled with
green-fluorescent Alexa Fluor® 488 streptavidin (Cat. no. S11223) and DNA was
stained with blue-fluorescent DAPI (Cat. no. D1306, D3571, D21490).
Cy3 NHS Ester
MW = 765.95
Properties
Excitation maximum (nm)
Emission maximum (nm)
Extinction coefficient (M-1 cm-1)
Quantum yield
Fluorescence lifetime (ns)
548
562
150 000 *
0.04 *
<0.3
(Note: The quantum yield of Cy3 is significantly
increased when in a rigid microenvironment, for
example in highly viscous solvents, or when
conjugated to biomolecules).
Upon attachment to biomolecules (for example
oligonucleotides), negligible shifts in excitation
and emission wavelength have been observed.
In addition no effects on these spectral
characteristics have been observed in the
presence of 10% DMSO or under a range of pH
conditions.
Excitation and emission spectra of
unconjugated Cy3 dye in phosphate
buffered saline
Cy5 NHS Ester
MW = 791.99
Properties
Excitation maximum (nm)
646
Emission maximum (nm)
664
Extinction coefficient (M-1 cm-1)
250 000 *
Quantum yield
0.27 *
Fluorescence lifetime (ns)
1.0
Upon attachment to biomolecules (for
example oligonucleotides), negligible
shifts in excitation and emission
wavelength have been observed.
In addition no effects on these spectral
characteristics have been observed in the
presence of 10% DMSO or under a range
of pH conditions.
Excitation and emission spectra of
unconjugated Cy5 dye in phosphate
buffered saline
Thiol-Reactive Fluorophores
The Thiol-reaction
The Thiol-reaction
For their participation in the process of reversible redox reactions, cysteine (Cys)
and homocysteine (Hcy同半胱胺酸) play crucial roles in physiological matrices.
Deficiency of Cys would lead to many diseases, such as hematopoiesis
decrease(造血作用), leucocyte loss (白血球), psoriasis (牛皮癬) etc. Hcy is a risk
factor for cardiovascular(心血管病變 ) and Alzheimer’s disease.
Some Cys and Hcy analyses have been developed in conjunction with HPLC,
capillary electrophoresis, immunoassay, colorimetric and fluorescence detection,
etc.
Bioconjugate Chem., Vol. 13, No. 3, 2002
Facile Synthesis of Thiol-Reactive Cy3 and Cy5 Derivatives
with Enhanced Water Solubility
lucifer yellow iodoacetamide,
dipotassium salt
5-iodoacetamidofluorescein
5-IAF
N,N'-dimethyl-N-(iodoacetyl)-N'-(7nitrobenz-2-oxa-1,3-diazol-4yl)ethylenediamineIANBD amide
•Org. Lett., Vol. 9, No. 17, 2007
A Thiol-Reactive Fluorescence Probe Based on DonorExcited Photoinduced Electron Transfer: Key Role of
Ortho Substitution
This result clearly supports the hypothesis that the closer the distance
between the electron donor (in this case, BODIPY) and the electron
acceptor (in this case, maleimide), the more strongly the fluorescence
will be quenched.
Fluorescence image of protein bands in SDS-PAGE
gel, obtained with a FluorImager595 (Amersham),
with excition at 488 nm.
Concentrations of 5, 10, 50, and 100 μg/mL BSA in
sodium phosphate buffer containing 5% DMSO as a
cosolvent (0.1 M, pH 7.4) were labeled with 30 μM
o-maleimideBODIPY at 37 °C.
After 60 min, the samples were treated with SDS and
β-mercaptoethanol, vortexed, and heated at 95 °C
for 5 min before being loaded on the gel for
electrophoresis.
Alexa Fluor® 488 C5maleimide
Oregon Green® 488
maleimide
A) BODIPY 499/508 maleimide
B) BODIPY FL N-(2-aminoethyl)maleimide
J. Am. Chem. Soc., 129 (34), 10322 -10323, 2007
A Highly Selective Fluorescence Turn-on Sensor for
Cysteine/Homocysteine and Its Application in Bioimaging
Absorption spectral changes of 1 (10 íM)
upon addition of Cys (0-330 íM). Inset
shows the color changes of 1 (10 íM) in the
presence and absence of amino acids and
peptide. From left to right, then bottom to
top: 1 (10 íM) and 1 with Ala, Cys, GSH,
Hcy, Glu, Try, Arg, Iso, Asp, Ser, Gly, Val,
Leu, Asn, Tyr, Met, Pro, Lys, Thr, and His,
respectively.
Two-photon fluorescence images
of PANC (a) and HeLa (b) cells
incubated with 10 μM 1 for 10 min
at 25 °C (λex = 880 nm).
B. Non-covalent (biological) Interaction
Policy and Chart
Ligands Binding
Fluorescence molecules
Specific target
Localization in vivo
Molecular modification
DNA or Protein binding
Biomarker or drug candidates
張健忠, 2007科學月刊6月號-螢光影像專輯2 (Figure credit: Sheh-Yi Sheu, Department of Life Science, NYMU)
Target Finding
Telomere:
The ends of the chromosomes .
Tandem repeats d(TTAGGG)n
3’ overhang single strand (100~200b).
Shorten in Normal cell,
remain constant in cancer cell
Telomerase:
The most important enzyme for telomere maintenance in
tumor cells so far!!!
Large (170kDa in man) RNA-dependent DNA polymerase
Over-express telomerase are found in 85% cancer cell
Telomerase:
How to degrade?
TTAGGG
AAUCCC
T
T
T
G
G
G
G
G
G
G
G
G
A
T
A
A
T
A
G
G
G
T
T
T
Different fluorescence
LD Hum24
C-C. Chang et al, J. Chin. Chem. Soc. 2003
1. Strong visible fluorescence.
2. The binding preference of BMVC is higher to quadruplexes than to duplexes.
Sensitivity assays (K~109)
Binding preference
Hum24
BMVC(0.5 µM)
2.5, 1, 0.5,0.25, 0.1, 0.05, 0.025, 0.01, 0.005 µM
C-C. Chang et al, Analytical Chemistry, 2003
USA patent:US6979738B2, 2005
C-C. Chang et al, Analytical Chemistry, 200
Mapping the localization of AP-G4
structures in metaphase chromosomes
Confocal microscopy, BMVC-stained
in metaphase chromosomes
1.85 ns
Color-coded image
C-C. Chang et al, Anal. Chem, 2006.
First evidence to find quadruplex structure in human.
Simple method for selfDiagnosis of cancer cells
BMVC
Self-diagnosis of cancer cells
AO
CL1-0 MRC-5 L1-0 MRC-5
C-C. Khang et al, The Analyst, 2007
United States Patent: APPLIED, 2007.
The detection limit is nearly to one single cancer cells.
Inoculation in nude mice
CL1-5 c/o
CL1-5 treated by
1 uM BMVC
1600
3
Tumor size (mm )
Model for inhibition of cell growth
1200
800
400
0
0
10
20
30
40
50
Time (day)
(Figure credit: Sheh-Yi Sheu, Department of Life Science, NYMU.
Other Generation SAR
D16
H24
BMVC C15 C18 C2
5uM 5uM 20uM 5uM
C11
C5 C12
27 BMVF
20uM 20uM 20uM 20uM 20uM
Binding preference to H24: BMVC, C2, C11
No Binding Preference: C15, C18, 27
Prefer Binding to Ld: BMVF
20 µM
SLR
(Structure localization relationship)
Nucleoli
Pyridinium side:
Increase efficient
Lysosome
Dimer:
take long time
Mitochondria
9-sub, flu enhance
N-position
Cell (2006) Vol. 125: 1137-1149
Ligand-Induced Dimerization of EGFR and Active and Inactive
States of Its Kinase Domain
Cell, Vol. 110, 775–787, September 20, 2002
EGFR is a rational
target for cancer
therapeutics
www.seas.upenn.edu
Efficient labeling of EGF and its intracellular delivery
Biomaterials, 28, 27, 3996
EGF binding sites
Normal lung cell
(HFL1) with NP-EGF
Lung cancer cell
(A549) with NP-EGF
EGFR
Labeled EGF
P
internalize
標靶分子療法:
抑制epidermal growth factor receptor(erlotinib and gefitinib)
platelet-derived growth factor receptor (imatinib and PTK787)
molecular target of rapamycin(temsirolimus and sirolimus)
farnesyltransferase (tipifarnib)
vascular endothelial growth factor receptor(PTK787)等單一藥物
A431 cells labeled with probes for histones and the epidermal growth factor (EGF)
receptor. Histones were detected with a mouse anti-histone antibody prelabeled
using the Zenon® Alexa Fluor® 488 Mouse IgG2a Labeling Kit (Cat. no. Z25102).
EGF receptors on the cell surface were detected with a mouse anti–EGF receptor
monoclonal IgG2a antibody prelabeled using the Zenon® Alexa Fluor® 555 Mouse
IgG2a Labeling Kit (Cat. no. Z25105).
Figure 1. A QD-EGF binds and activates cells expressing a green fluorescent protein
(GFP)-fused EGFR. (a) Red QD-EGF and green GFP-EGFR colocalize on the cell
membrane within minutes and (b) are rapidly internalized together into endosomes in
the cytoplasm. (c) Individual receptors on long sensory filaments called filopodia can
be visualized by QD-EGF binding. Images are single confocal sections through living
cells.
Nanotechnology 2006
Biomedical Optics & Medical Imaging 2008
•
Metals are essential for sustaining all forms of life, but alterations in their cellular
homeostasis are connected to severe human disorders, including cancer, diabetes
and neurodegenerative diseases.
•
Fluorescent small molecules that respond to metal ions in the cell with appropriate
selectivity and sensitivity offer the ability to probe physiological and pathological
consequences of the cell biology of metals with spatial and temporal fidelity.
•
Molecular imaging of normal and abnormal cellular metal ion pools using these new
chemical tools provides a host of emerging opportunities for visualizing, in real time,
aspects of metal accumulation, trafficking, and function or toxicity in living systems.
•
This review presents a brief survey of available synthetic small-molecule sensor types
for fluorescence detection of cellular metals.
•
Designing metal-responsive fluorophores for cellular use
•
Small-molecule fluorophores for cellular zinc
•
Fluorescent sensors for cellular copper
•
Synthetic fluorescent reporters for iron
•
Fluorogenic probes for toxic heavy metal detection-Mercury, Lead and cadmium.
Metallic Fluorescent Sensor
J. AM. CHEM. SOC. 2007, 129, 1500-1501
597 nm Ф= 0.59
656 nm Ф= 0.12
1 (5 íM) in the presence of
different metal ions (150 μM)
in Tris-HCl (0.01 M) solution
(acetone/water ) 9/1,
v/v, pH 7.4)
Physiologically important metal ions which exist in living cells, such as Ca2+, Mg2+, Na+,
K+, and Fe3+, do not give any responses at 30-fold excess concentration. Most heavy
and transition metal ions, such as Hg2+, Ag+, Mn2+, and Zn2+, also have no interference.
Only Cr3+ induces very slight fluorescence enhancement. Ni2+ and Cu2+ obviously
quench the fluorescence to some extent, which always meet in the other metal ion
sensors.
Photos by the Naked Eye and Fluorescence of 1 with Cd2+ and Zn2+
Visible emission observed by both naked
eye and fluorescence from samples. The
concentration of 1 was 5 µM, and the
concentration of Cd2+ and Zn2+ was 250 µM.
The first example of fluorescent Cd 2+ sensors which can distinguish Cd 2+
from Zn 2+ with both emission shift and fluorescence intensity.
-CuCl2
Org. Biomol. Chem., 2006, 4, 1966 - 1976
+CuCl2
Cu+2
J. Am. Chem. Soc. 2006, 128, 10-11
J. Am. Chem. Soc., 125 (6),
1468 -1469, 2003.
K+1
Prof. Koji Suzuki (Keio)
Creation of Nanochenical Probes
生醫奈米影像技術
Biomedical Molecular Imaging
VI. Fluorescent Organic Nanoparticles
Formation in Lysosomes for Cancer
Cell Recognition
Cheng-Chung Chang
• Introduction :
Organic nano particle ,FON , Identify of FON
• Molecular Design:
Visible emission, pH dependent, Aggregation
• FON as cellular probe
AIEE (Aggregation-Induced Enhanced Emission)
• Other applications
Nanoparticles
Organic Nanoparticles
natural
nanoparticles
man-made
nanoparticles
cell components
active substances
(pharmaceuticals)
viruses
pigments
environmental
colloids
(on surfaces, in
water, in air)
polymer dispersions
Fluorescent Nanoparticles
Conor Hogan (Department of Chemistry,
La Trobe University)
micellar systems
Supermolecules and
dendrimers
Aqueous forms of application
Luminescence property
Protein aggregates
(as nuclei for crystals
Angew. Chem. Int. Ed. 2001, 40, 4330 - 4361
FON
Methods for the preparation of nanoparticles
Angew. Chem. Int. Ed. 2001, 40, 4330 - 4361
Fluorescent Organic Nanoparticles (FONs)
Douglas C. Neckers
(Org. Lett., Vol. 10, No. 2, 2008)
Soo-Young Park
(J. AM. CHEM. SOC. 9 VOL. 124, NO. 48, 2002)
Soo-Young Park
Angew. Chem. Int. Ed. 2007, 46, 1978 –1982
Cellular Image
Co-culture normal and cancer cell line
Bright spots present in cancer cell but not normal cell
1. Fluorescent bright spots.
2. Cytoplasm of cancer cell.
3. Normal cell.
Mean Intensity
300
1
2
3
200
100
0
450
500
550
nm
600
650
Identification of FONs
Emission spectra studies in THF/ H3O+ solvent pairs
Transmission Electron Microscopy (TEM)
pH~ 4.21
pH ~3.95
Quantum Yields (Φ)
100
a)
0.3
0.2
75
75
0%
H
N
b)
H
N
100%
75%
50%
25%
0% THF
100
0%
0.3
S
S
N
N
PVP
N
BPVP
0.2
0.1
0.1
0.0
0.0
500
600
700
800 500
600
700
800
Wavelength (nm)
04
04
Aggregation-Induced Enhanced Emission
(AIEE)
The nanoparticles are easily observed in TEM images with 5~ to 100 μΜ in H3O+
/THF mixed solutions.
The AIEE areas are evaluated (considering the dissociation ability of HCl in the
THF solvent), cover the acidic condition of lysosomes of living cells.
Quantum Yields (Φ)
0.4
c)
0.4
d)
100% THF
75%
0%
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
3.0
pH
3.5
4.0
4.5
5.0
5.5
6.0
6.5
Either in organic or aqueous solutions, the fluorescent intensities of PTZ derivatives
are decayed upon protonation.
The neutral water/THF mixed solutions, PTZ derivatives do not exhibit AIEE.
The protonated PTZ serve as surfactant-like molecules due to their amphiphilic
characteristics.
H+
N
+HN
Conductivity (mV)
0.9
Hydrophilic
NH +
+HN
NH
S
S
0.6
NH S
NH
NH
S
H
N
+
0.3
N
H
-5
BPVPH (CMC=2.1x10 M)
+
-5
PVPH (CMC=1.2x10 M)
NH +
S
Hydrophobic
S
+HN
S
HN
HN
S
HN
S
0.0
0.0
50.0
100.0 150.0 200.0 250.0
NH +
[conc of PVP or BPVP, uM]
N
H+
NH +
Emission Spectra in Cell
Solvent effect of protonated molecule
Protonated PTZ aggregate in the less polar vacuoles
to form FONs
a)
Toluene
EA
THF
CHCl3
0.2
Toluene
EA
CHCl3 DMSO
DMSO
300
Mean Intensity
Quantum Yields (Φ)
0.3
0.1
0.0
500
600
700
800
Waveleng
1
2
3
200
100
0
450
500
550
nm
600
650
Model and Mechanism
Incubate PTZ
Protonation
Cytoplasm (fluorescence quench)
Lysosomes
vacuoles
Fluorescent Organic
Nanoparticles (FONs)
Aggregation-Induced
Enhanced Emission (AIEE)
THF / H3O+ ( pH~ 4.21)
vacuoles
Lyso Tracker
FONs / Lyso Tracker
100 % 75 %
0%
Cationic FONs
FONs form in acidic aqueous.
AIEE reach more than 100 folds. (in H3O+/THF w.r.t neutral monomer in aqueous)
Recognize cancer cell from normal cell.
500
pH~7.0
pH<5.5
400
300
100% 25%
498
467
200 nm
N
200
x20
100
0
400
500
nm
600
H+
THF
0%
Anionic FONs
H
N
S
HO
OH
FONs form in alkaline aqueous.
AIEE reach more than 60 folds. (in OH-/THF w.r.t neutral monomer in aqueous)
200
H2O
OH
150
506
N
100
OHN
50
0
400
520
x5
500
S
HO
OH
H
N
nm
600
700
S
OCH3
FON as colorimetric sensor
Jinsang Kim
Adv. Mater. 2008, 20, 1117–1121
Haibing Li
Tetrahedron Letters 49 (2008)
7438–7441