单分子技术原理与应用
北京大学
生物医学前沿创新中心(BIOPIC)
单分子与活细胞动态过程实验室
孙育杰
sun_yujie@pku.edu.cn
应用单分子超分辨荧光研究细胞动态过程和精细结构
发展单分子荧光技术研究细胞动态过程
Sun & Goldman, 2011
Sun et al,2007
Sun et al., 2009
Sun et al., 2011
In vitro reconstitution of cellular membrane deformation
应用单分子超分辨荧光研究细胞动态过程和精细结构
超分辨显微技术研究细胞超微结构及功能
10大前沿生物技术方法
2014年10月 Nature Methods
创刊10周年特刊
单分子荧光和超高分辨率荧光显微技术
• Biological questions that motivate the development
of single molecule techniques.
• Brief historical review of single molecule techniques.
• Single molecule imaging/spectroscopic techniques.
• Single molecule force/manipulation techniques.
• Biological applications of single molecule techniques.
• Super-resolution microscopy
Cell: a chaotic/hectic yet organized world
David C. Goodsell, Scripps Institute
Cell: a chaotic/hectic yet organized world
Lipid droplets trafficking in embryo cells
From Internet
Mitosis
From Internet
Melanosome Movement
John A. Hammer, III, NIH
Cytoplasmic streaming in plant cells
From Internet
为什么细胞生物学和分子生物学需要单分子技术
眼见为实 ---- 在细胞生物学领域，单分子研究的终极目标之一就是在体内实时观测
单分子，包括生物大分子的运动并以此在分子水平上理解生理条件下的细胞过程
The wonders of a tiny cell
Cytoskeleton and motor protein dynamics
活细胞内过程需要单分子观察
Molecular Motors drive many processes in the cell
• Polymerization motors:
Actin, Microtubule
• Cytoskeletal motors:
Myosin, Kinesin, Dynein
Kinesin
Myosin
actin
Dynein
Green = microtubule
Red = actin
microtubule
Pollard & Earnshaw, Cell Biology, 2nd ed.
• Rotary motors:
FoF1-ATP synthase
The bacterial flagellum
Dimroth et al. EMBO, 2006
Credit: Fuller, N.R., NSF
• Nucleic acid motors:
RNA & DNA polymerase,
Helicase, etc.
Johnson et al. Cell, 2007
Wang et al. Nucleic Acids Res., 2004
什么是单分子技术
研究对象
技术手段
应用范围
单一小分子、生物大分子和分子复合体/聚
合体
观察
在纳米空间尺度和
毫秒时间尺度上精
确测量单个分子的
距离、位置、指向、
分布、结构以及各
种动态过程
操纵
在皮牛到纳牛力学尺
度上操纵和检测单个
分子的力学行为和动
态过程
单分子技术简要分类
光谱/荧光
力学操纵
荧光 Fluorescence
• Optical tweezers
• Magnetic tweezers
• Scanning probe microscopy
• TIRF: Total-internal reflection fluorescence
• SMFP: Single molecule fluorescence polarization
• FRET: Förster resonance energy transfer
• FLIM: Fluorescence lifetime imaging microscopy
• Confocal microscopy
• Two-photon microscopy
荧光超分辨 Fluorescence Super-resolution
• STORM: Stochastic optical reconstruction microscopy
• PALM: Photo-activated localization microscopy
• STED: Stimulated emission depletion
• SIM: Structured-illumination microscopy
光谱 Spectroscopy
• SRS: Stimulated Raman Scattering
• FCS: Fluorescence correlation spectroscopy
……
……
• Electron microscopy
• Tethered particle motion (TPM)
……
单分子测量技术的优点除了可以提供与集合系统
测量相近的结果，还体现在下述几点
（1）超高灵敏度和超小的样品用量；
（2）得到的是实际几率分布，而不是平均值；
（3）测量单一分子及其相互作用的差异性和多样性；
（4）实时直接观测单个分子的反应动力学路径；
（5）观察分子复合体形成、解离等动态过程；
（6）可以捕捉单分子随机过程和分子构象变化的中间态；
（7）可以测量稀发但重要的信号和分布，而在集合系统测量时，这些
事件通常都被主要信号掩没；
（8）可以测量非平衡态和不同步的体系。很多的细胞分子生物学过程
都是这样的体系，比如分子移动和转动。
单分子技术对生物基础研究非常重要
Diverse views of
VDAC structures and
functioning
In the field of Cell Biology:
advancement has been
made in molecular
motors, protein folding,
305
replication, transcription,
and translation
as well
Emerging Single
Molecule
as some
exciting new
Techniques I
tools “single molecule
genome sequencing”
and “single molecule
immunoprecipitation”.
The occurrence in the scientific literature of papers on single-molecule methods. The number is
calculated from a PubMed search displayed as a function of the year from the 1980s until the end
of 2006. A Survey of Single-Molecule Techniques in Chemical Biology, Cornish and Ha, ACS
Chem. Biol., 2007
概要
• Biological questions that motivate the development
of single molecule techniques.
• Brief historical review of single molecule techniques.
• Single molecule imaging/spectroscopic techniques.
• Single molecule force/manipulation techniques.
• Biological applications of single molecule techniques.
• Super-resolution microscopy
Richard Feynman's talk at the 1959
meeting of the American Physical
Society at Caltech
Detecting Single Molecules
Ion channels < ----- > patch clamp
Pioneer Work
Nobel Prize in Physiology or
Medicine in 1991
Erwin Neher
Bert Sakmann
Detecting Single Molecules
Pioneer Work
最早用光学手
段检测单分子
的工作
Detecting Single Molecules
Daniel Axelrod 于1981年发明了TIRF – 完全内反射荧光显微镜技术
Pioneer Work
Detecting Single Molecules
Pioneer Work
其后Moerner和Kador在1989年在低温下实现了用光学手段观测单分子
1990年，Orrit 和 Bernard 也用光学手段实现了单分子观测
Detecting Single Molecules
Pioneer Work
Eric Betzig在1993年用近场光学显微
镜实现了室温下对单分子的观测
Sunney Xie在1998年在生理环境下用
单分子荧光显微镜研究酶学
Detecting Single Molecules
Pioneer Work
Taekjip Ha在1996年实现了单分子FRET
Toshio Yanagida 于1997-2000年发展了基于物镜的
TIRF技术并首次成功应用于活细胞内单分子成像
Manipulating Single Molecules
Pioneer Work
光镊 Optical Trap
Arthur Ashkin 于1970年发明了光镊 （Optical Trap/Optical Tweezers）
In 1987, Arthur Ashkin and Joseph M. Dziedzic demonstrated the first application of the
technology to the biological sciences, using it to trap an individual tobacco mosaic virus and
Escherichia coli bacterium
Manipulating Single Molecules
光镊 Optical Trap
1997 Nobel Prize in Physics
光镊束缚和控制单个原子
Pioneer Work
Manipulating Single Molecules
Pioneer Work
光镊 Optical Trap
Since 1990, Optical tweezers have been particularly successful
in studying a variety of biological systems
Carlos Bustamante
Dynamics and
Forces of molecular
motors
James Spudich
Steven Block
Pioneer Work
Manipulating Single Molecules
磁镊 Magnetic Tweezers
A magnetic tweezer is a scientific instrument for exerting and measuring forces on
magnetic particles using a magnetic field gradient.
David Bensimon
Vincent Croquette
Nynke Dekker
Neuman & Nagy, Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic
force microscopy, Nat Methods, 2008, 5(6):491
Manipulating Single Molecules
Pioneer Work
原子力显微镜 Atomic Force Microscopy
The precursor to the AFM, the scanning tunneling microscope, was developed
by Gerd Binnig and Heinrich Rohrer in the early 1980s
Nobel Prize for Physics in 1986
Binnig, Quate and Gerber invented the first atomic force microscope
(also abbreviated as AFM) in 1986
概要
• Biological questions that motivate the development
of single molecule techniques.
• Brief historical review of single molecule techniques.
• Single molecule imaging/spectroscopic techniques.
• Single molecule force/manipulation techniques.
• Biological applications of single molecule techniques.
• Super-resolution microscopy
Single cell gene expression (mRNA)
analysis using single molecular
resolution fluorescence imaging in
fixed and living cells
1959
40 years
•
•
•
•
Zenklusen D, et al. Single-RNA counting reveals alternative
modes of gene expression in yeast. Nat Struct Mol Biol 2008
Raj A, et al. Stochastic mRNA Synthesis in Mammalian Cells.
PLoS Biol 2006.
Golding I, et al. Real-Time Kinetics of Gene Activity in
Individual Bacteria. Cell 2005
Chubb JR, et al. Transcriptional Pulsing of a Developmental
Gene. 2006
Study of Single Molecules used
to be very hard
Small signal
Large noise
Light
Current
Force
…
Background noise
Thermal noise
Circuit noise
…
S/
N
单分子检测的技术难点
• 一个荧光分子在光漂白前大约可发射 300,000个光子。
• 检测效率: Objective (30%)´ Dichromatic mirror (85%)´
Barrier filter (80%)´ Detector (50%) = 10%
Room light = 5x107 photons/μm2/s
• 系统噪音：dark current, readout noise, shot noise etc.
• 样品噪音：
(1) Substrate和光学器件的荧光
(2) 其它荧光分子的荧光
S/N 与单分子定位精度
光学衍射
Diffraction-limited Spot
Point Spread Function （PSF）
体外量子点
体内荧光蛋白
Improvement in all aspects of contemporary microscopy
makes Single Molecules eventually VISIBLE
Microscope
Light sources
(illumination/excitation)
Magnification/Objectives
Detectors (Camera etc.)
Stages/Displays …
Stage: less vibration
and drift
单分子荧光显微镜基本配置
显微镜
物镜
激光
研究级倒置
荧光显微镜
Olympus 100x
1.4NA Oil
UPlanSApo
405 nm (~50 mW)
for photoactivation
of most
fluorophores
Nikon 100x
1.49NA Oil
Optical
modulation
AOTF
TIRF-level flatness
405/488/561/647 polychroic mirrors
相机
EMCCD
Quad-band emission
filters.
561 nm laser
(~100 mW) for
EosFP or Cy3B
647 nm (~200 mW)
for Cy5/Alxea647.
Filter/
Splitter
Mechanical
shutter
sCMOS
Nikon Ti-E
Dual-view or Quad-view
ND filters
Polarizor/Wave
plate
TTL modulation
Laser
Conventional Light Sources
Laser
Magnification/Objectives
• Larger collection of light
• Higher transmission
• Less aberration
Detectors: Electron Multiplied Charge Coupled Device
(EMCCD) Cameras
S/N vs. EM gain
S/N
Single Photon Sensitivity:
• Higher Quantum efficiency
• Lower Dark Current
• Higher amplification
EM gain
Fluorescence Probes: Organic dyes
Fluorescence Probes: Fluorescent Proteins
“A protein giving solutions that look slightly greenish in sunlight though
only yellowish under tungsten lights, and exhibiting a very bright, greenish
fluorescence in the ultraviolet of a Mineralite [a handheld ultraviolet lamp],
has also been isolated from squeezates”
---- Shimomura et al. 1962
Fluorescence Probes: Quantum Dots
Highly fluorescent nanometer-sized single crystals semiconductor materials
量子点
如何评价一个荧光基团
Extinction coefficient/Absorption cross-section
Quantum yield
Photostability
Brightness
比较三种荧光探针: 有机染料、荧光蛋白和量子点
• Size: if it matters for target proper functions. [Organic dyes]
• Toxicity: if it poisons cells or animals. [Fluorescent proteins]
• Brightness: extinction, quantum yield. [Quantum dots]
• Photostability: photobleach, quench, blinking. [Quantum dots]
• Labeling specificity: approaches to targeted labeling. [Fluorescent
proteins]
• Environment sensitivity: pH, ionic, temperature etc. [?]
降低背景噪音的单分子荧光技术
降低背景噪音的单分子成像手段
Ø Light-sheet荧光
显微镜：适用于细胞内
部，如细胞核内的单分
子过程.
Ø 双光子或共聚焦荧光
显微镜：常用于较深较大
的组织样品，可用于细胞
内部，如细胞核内的单分
子过程.
Ø TIRFM –– 全内反射
荧光显微镜：限于较薄
的细胞和细胞区域.
𝑆
𝑁
TIRFM –– Total Internal Reflection Fluorescence Microscopy
Snell’s law
n1 /n2 = sin(θ2)/sin(θ1)
Beads Fluorescence Intensity vs. Piezo Mirror
Votage
4.0E+05
water
n2 = 1.330
Intensity (A.U.)
Critical
Angle
3.5E+05
3.0E+05
V Increase
V Decrease
2.5E+05
2.0E+05
1.5E+05
1.0E+05
5.0E+04
glass
n1 = 1.513
Total Internal
Reflection
Nikon Microscope Website
0.0E+00
1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50
Votage (V)
Cytoskeletal actin polymerization and branching
Epi
Blanchoin et al., (2000) Nature, 404:1007-1011
TIRF
Sun Y. et al., Curr. Biol. 2013
Light sources
(illumination/excitation)
Magnification/Objectives
Detectors (Camera etc.)
Stages/Displays …
Fluorescent labeling
Imaging mode
Single Molecules Imaging
概要
• Biological questions that motivate the development
of single molecule techniques.
• Brief historical review of single molecule techniques.
• Single molecule imaging/spectroscopic techniques.
• Single molecule force/manipulation techniques.
• Biological applications of single molecule techniques.
• Super-resolution microscopy
单分子技术在体外的应用
单分子技术在体内的应用
Nucleosome
Membrane
Helicase
Gyrase
Focal adhesion
Lac repressor
RNA polymerase
Molecular motors
Splicesome
NPC
Transcription
Ribosome
mRNA
Cytoskeletal motors
Virus tracking
Flagellar motor
F-type ATP synthase
复制
转录
RNA转运
剪切
翻译后处理/修饰
翻译
蛋白转运/分布
Ion channels
Calcium pump
蛋白-蛋白相互作用 信号响应—信号传导
EGF receptor
离子通道
Ras
Peters, Annu. Rev. Biophys. Biomol. Struct. 2007
转录调控
能量过程（线粒体） …….
单分子技术研究DNA別构效应
Direct observation of Binding and Unbinding
別构效应
Allostery = “allos” (other)+ “stereos” (object)
当效应因子结合到远离活性中心的变构位点处能够通过蛋白质的长程构象变化
来影响蛋白质活性中心功能（酶活性）的现象
koff
koff
可能的功能：转录调控？
http://202.204.115.67/jpkch/jpkch/2008/wswx/
koff of LacR is modulated by binding of T7 RNAp
as a function of their separation
Science, 2013
体外
实验
单分子荧光实验研究
DNA別构效应
LacR
L
T7RNAp
t
N (t ) = A exp(- )
t
10 µm
Average binding time = t
Standard error = t /sqrt(N)
koff = 1/ t
Science, 2013
koff(saturating T7 RNAp) / koff(no T7 RNAp)
STABILITY OF LACR AS A FUNCTION OF SEPARATION FROM T7
1.6
If DNA is nicked
If DNA has mismatched bases
1.4
LacR dissociates faster
1.2
1.0
0.8
LacR binds longer
0.6
0.4
0
5
10
15
20
25
30
35
Separation, L (bp)
40
45
50
Science, 2013
核小体影响其附近转录因子DNA
结合强度的实验结果
Science, 2013
单分子技术研究马达蛋白
Direct observation of Motion
FoF1-ATPase (ATP合成酶)
位于线粒体内膜基质一边，由F0和F1构成的复合体。是一种ATP驱动的质
子运输体，当质子顺电化学梯度流动时催化ATP的合成；当没有氢离子梯
度通过质子通道Fo时，F1的作用是催化ATP的水解。
细胞骨架分子马达
• Polymerization motors:
Actin, Microtubule
• Cytoskeletal motors:
Myosin, Kinesin, Dynein
Property
Fact
Hydrolyze ATP
~ 20 kBT
cycle time
diffusion coeff.
~ 20 ms
~ 10 nm (head)
~ 100 nm (overall)
~ 10 µm2/s
stiffness
~ 1 pN/nm
force
~ 5 pN
speed
from 0.5 to 30 µm/s
size
Molecular Motor Toolbox
ELC
Myosin V
Calmodulins
37
nm
5.5
nm
Actin
Filament
Motor
Domain
Coiled Coil
Cargo Binding
Domain
Molecular Motor Toolbox
ELC
Myosin V
Calmodulins
37
nm
Coiled Coil
Cargo Binding
Domain
Motor
Domain
Microtubule
8 nm
5.5
nm
Kinesin-1
Actin
Filament
Coiled Coil
Cargo Binding
Domain
Motor
Domain
Molecular Motor Toolbox
ELC
Myosin V
Calmodulins
37
nm
Coiled Coil
Cargo Binding
Domain
Motor
Domain
Microtubule
Cytoplasmic
Dynein
IC Dynactin
Binding
Domain
5.5
nm
Microtubule
Binding Stalk
LIC
8 nm
IC
Actin
Filament
Kinesin & Dynein in Neuron
MOLECULAR MOTORS AND MECHANISMS OF
DIRECTIONAL TRANSPORT IN NEURONS
Hirokawa and Takemura NATURE REVIEWS
NEUROSCIENCE 2005, 6:201
The Myosin Superfamily
The Myosin Superfamily
The Myosin Superfamily
Cytoplasmic streaming in plant cells
Myosin
X
Myosin
V
Lipid droplets trafficking in embryo cells
Myosin VI
Myosin X
Helical Pitch = 72 nm
Adapted and modified from Rock et al., PNAS (2001) and Krendel & Mooseker, Physiology (2005 )
In vitro motility assays are used to measure motor activity
Toyoshima et al. (1987) Nature 328:536-539.
Myosin V Strolling
Single Molecule
Study of Molecular
Motors
Myosin Stepping Mechanisms and Dynamics by Single
Molecule Position and Orientation Measurements
Sun et al., 2007
Sun et al., 2010
Ø
Width of l/2 » 250 nm
Diffraction limited spot
Prism-type TIR 0.2 sec integration
center
280
240
Photons
200
160
120
width
80
40
0
5
10
15
Y ax
is
20
Z-Data from Columns 1-21
photon pixelation
noise
background
noise
With enough photons (signal to noise)
…
Center can be determined to » 1 nm.
Yildiz et al., 2003, Science
20
25
25
15
X Da
10
ta
5
0
measurements of myosin motor stepping
Streptavidin-QD
120
NEMMyosin
Biotin
Tag
Cover glass
Y pixels
118
116
114
404
406
408
410
412
414
X pixels
800
120
600
500
y position (nm)
118
Y pixels
400
300
200
116
100
0
100
200
0
2
4
6
8
10
12
114
404
14
Time (s)
Time
(s)
406
408
410
x position
(nm)
X pixels
412
414
800
700
600
500
t (nm)
Displacement (nm)
Displacement (nm)
700
400
Sun, 2010, Nat Struct Mol Biol
measurements of myosin motor stepping
QD labeled
labeled Myosin
Myosin XX motors
motors
QD
Label Position
Position 11
Label Position 2
Label
short
P(t) = ke−kt
long
<Dwell time> = 0.89 s
P(t) = tk2e−kt
<Dwell time> = 1.62 s
0
Super
long
Sun, 2010, Nat Struct Mol Biol
Ø Precise localization proves single myosin motors step hand-over-hand
short
long
Differential labeling of myosin V heads with quantum dots allows
direct visualization of hand-over-hand processivity
Warshaw, D.M. et al. Biophys. J. 88, L30–L32 (2005).
Correlation between the
movement of MyoV-HMM
and the binding
/dissociation of deacaminonucleotide. Images of
Alexa-Fluor-568–MyoV-HMM
and deac-aminoATP
fluorescence were acquired
simultaneously with a DualView system.
Ø Kinetics features of myosin motors
Trailing
head
Leading head
Vale, J. Cell Biol., 2003
Coordination between the two legs of Myosin V
An Optical Trap study
Deterministic Working Stroke
Thermal Motions
This image cannot currently be displayed.
Completion of Step
单分子体内实验应用
3D movement of GLUT4-containing
vesicles in living adipocytes
Sun et al., Nano Lett, 2009
Next Level:
Ex vivo Single Molecule
体外单分子荧光实验研究分子马达驱动的膜形变
lysosome
Kinesin 1
Autolysosome
Microtubule Network
Mitochondria
500 nm
In collaboration with Li Yu, Tsinghua University
Membrane deformation in the cell -- Two model systems
Autophagic Lysosome Reformation (ALR)
1. Mechanism for budding or fission of tubules
and proto-lysosome?
2. Curvature formation and maintenance?
3. Sorting mechanism for cargo segregation
and membrane separation?
Yu et al, Nature, 2010
Mitochondrial Network Formation
1. Fission and fusion?
2. Morphology and functional states?
In vitro and In vivo
Filter paper
Slides
DoubleSide Tape
Pipette tips
Flow
Coverslip
Controllable ingredients
Controllable concentration
Low background
Physiological
In vitro Reconstitution
lysosome
Kinesin 1
P
PE Biotin
P
PIP2
PE Rhod
Autolysosome
Microtubule Network
AP2
K560 Biotin
Clathrin
Full Length Kif5b
Mitochondria
Fluorescent
Microtubule
500 nm
Tubulin
Antibody
In vitro Reconstitution
体外重构 （In vitro reconstitution）
Autolysosome
Mitochondria
Working model
Coordination?
Deforming
Concentrating
Recruiting
Budding
Kenisin
Clathrin
AP2
PIP2
Remaining questions
1. Fission of tubules and proto-lysosome --- Dynamin ?
2. Sorting mechanism for cargo segregation and membrane separation
--- PI4P, PI5K1, PI(4,5)P2 , Clathrin, and Curvature?
概要
• Biological questions that motivate the development
of single molecule techniques.
• Brief historical review of single molecule techniques.
• Single molecule imaging/spectroscopic techniques.
• Single molecule force/manipulation techniques.
• Biological applications of single molecule techniques.
• Super-resolution microscopy
生命科学对高分辨率
显微成像技术的需求
细胞的尺度
~0.02mm
人眼的分辨率
~0.1mm
光学衍射极限与生物超高分辨率成像的需求
细胞骨架
荧光显微镜图像
细胞骨架
细胞骨架
电子显微镜图像
Green = microtubules
Red = actin
Medalia et al. Science, 2002.
Abbe衍射极限
分辨极限
无法分辨
可分辨
爱里斑
爱里斑
~ 300 nm
1873年，德国科学家Ernst Abbe
提出“光学衍射极限理论”。
电磁波波长范围和光学显微镜空间分辨率
波长
≈ λ/2 ≈ 300 nm
数值孔径和光学显微镜空间分辨率
数值孔径 NA = n*sin α
折射率 n
折射率 孔径角
光学衍射极限与生物超高分辨率成像的需求
荧光显微镜
电子显微镜
哺乳动物细胞
蛋白质
核糖体
病毒
细胞骨架
线粒体
光学显
微镜
分辨率
300nm
• 尺度：在细胞内，很多亚细胞结构、细胞器和生物大分
子的尺寸都在微米和几十纳米的级别。
• 浓度：细胞内非常拥挤，1 μM 的浓度相当于 1 μm3中
有 ~600 个分子，也相当于衍射极限体积内有 5个分子。
细菌
细胞骨架超高分辨率
荧光显微镜图像
2014年诺贝尔化学奖
“突破”衍射极限
超高分辨率显微成像技术
突破衍射极限的超高分辨率光学成像
突破衍射极限的超高分辨率光学成像
Nature:2008年度方法
超高分辨率荧光成像
Marta Fernández-Suárez and Alice Y. Ting, Nature Review: Mol. Cell Biol. 2008
突破衍射极限的超高分辨率光学成像
活细胞超高分辨率
RESOLFT 显微镜
S. W. Hell: "Diffraction-unlimited all-optical imaging and writing with a
photochromic GFP" Nature 478, 204 – 208 (2011)
S. W. Hell: "Nanoscopy of Living Brain Slices with Low Light Levels"
Neuron 75, 992 – 1000 (2012)
S.W. Hell. "rsEGFP2 enables fast RESOLFT nanoscopy of living
cells“ eLIFE (2012)
S.W. Hell. "Nanoscopy with more than 100,000 ‘doughnuts’ ” Nature
Methods (2013)
突破衍射极限的超高分辨率光学成像
2014， 2015
活细胞超高分辨率显微镜
lattice light sheet based NL-SIM
Bi-chang Chen … Eric Betzig, Science 2014
Dong Li … Eric Betzig, Science 2015
“突破”衍射极限和实现超高分辨率成像的基本思想
任何光源聚焦成的光斑的大
所有分子同时发光，其“爱
小也是受衍射极限限制的！
里斑”重合，导致无法区相
邻分子。
基本思想一
用小于衍射极限的
光斑来扫描样品
缩小照明光斑点
扩散函数 + 扫描
控制每次“可见”的分
子数目，使得相邻分子
不同时发光
单分子定位
+重构
基本思想二
Single molecule localization microscopy
基于随机重构的突破衍射极限的超高分辨率光学成像
STochastic Optical Reconstruction Microscopy (STORM)
Photoactivated Localization Microscopy (PALM)
From FIONA to Super-Resolution Imaging
利用光控的荧光基团进行随机重构
Imaging
Laser
光控荧光基团
Photoactivable
Photoswitchable
Photoconvertable
利用光控的荧光基团进行随机重构
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
精确的单分子定位
荧光强度
X 轴 （像素）
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
Weak
Activation
Laser
利用可激活的荧光基团进行
随机重构
Strong
Imaging
Laser
利用可激活的荧光基团进行
随机重构
利用可激活的荧光基团进行
随机重构
“突破”衍射极限和实现超高分辨率成像的基本思想
STED和STORM/PALM技术的对比
超高分辨率光学成像技术简要比对
技术名称
空间
分辨率 (nm)
STED
STORM
PALM
SIM
NSIM
RESOLFT
SOFI#
µ n
3B
40-60
10-20
20-30
100
70
40-60
(up to 4)
~80 nm
~50
(扫描区域
100x100μm2)
~10 s
~1 s
~30 s
~10 s
~1 s
~1 s*
1-4 s
~4 s
探针
主要为有机
染料
时间分辨率
其它
使用较强激
光，可能伤
害生物样品
饱和原理
有机染料， 荧光蛋白，
都可以
不易做活
适于活细
细胞
胞
•
•
•
最高z轴分辨率
可定量Couting
单分子追踪
最相似于
传统显微
镜，简单
易用
单分子定位原理
干涉
荧光蛋白， 荧光蛋白， 适合用量
子点
适于活细 适于活细
胞
胞
对荧光蛋
白光稳定
性要求高
对荧光蛋
白光稳定
性要求高
饱和原理
对SNR要
求低，基
于算法，
高阶可能
有假象
需抗漂白且
闪烁多
对SNR要求
低，基于算
法，计算量
巨大
荧光涨落原理
* 基于平行化的扫描方式
#: Super-resolution optical fluctuation imaging (SOFI) achieves resolution enhancement based on the statistical analysis of temporal
fluorescence fluctuations.
Nucleoli
Nucleus
Endoplasmic Reticulum
Mitochondia
Many biological
structures and
processes in cell
biology, microbiology
and neurobiology
Plasma Membrane
Golgi
Microtubule
Lipid Rafts
Peroxisomes
Cytosol
Actin
Lysosomes
RESOLFT 实验数据
Live-cell imaging with parallelized RESOLFT nanoscopy
S.W. Hell. "Nanoscopy with more than 100,000 ‘doughnuts’ ” Nature Methods (2013)
STORM超分辨显微镜解析神经细胞突触区分蛋白分子分布
Adish Dani, Bo Huang, Joseph Bergan, Catherine Dulac, and Xiaowei Zhuang Neuron, 2010
双物镜STORM解析神经细胞轴突中Actin周期结构
超高分辨率显微技术的发展趋势
从显“微”镜时代带入到显“纳”镜时代
•
更高： 更高的空间分辨率
•
更快： 更快的时间分辨率
•
更深： 更深的成像深度
活体研究
超高分辨率显微技术的发展趋势
•
原理创新： 发展新型的超分辨技术和标记技术
高速超分辨：超快扫描元件，高灵敏检测元件等
深层超分辨：自适应光学，组织透明化（CLARITY）等
•
发展和优化荧光探针：
优化现有探针：亮度、光稳定性、转换速率以及发射波长等
开发新型探针：上转换纳米材料，荧光钻石，石墨烯量子点等
•
模态融合： 没有哪种技术能兼顾时间分辨率、空间分辨率、空间尺度。对于超分辨技术而
言，至少有三种值得融合的技术。
双光子荧光显微镜：成像深度深
电子显微镜：即光电融合显微技术CLEM (Correlative Light&Electron Microscope)
片层光 light-sheet： 2014年Nature Methods年度方法
•
高效的图像处理软件和算法：超高分辨率动态成像以及大样品成像
Seeing what’s going on in
the cell is very fun!
谢谢！
单分子技术和应用的书
推荐
Phil Nelson
Rob Philips
UPenn
Caltech