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Fluorescence Nanoscopy
Microscopy
Lasers
Optics 高甫仁高甫仁高甫仁高甫仁& Collaborators陽明大學生醫光電工程研究所陽明大學生醫光電工程研究所陽明大學生醫光電工程研究所陽明大學生醫光電工程研究所July 23, 2010
Outline
�A brief overview on optical microscopy
�Toward optical super resolution __ Abbe’s diffraction limit and nanoscopy
�Fundamentals and opportunities of FLIM/FRET
�Visualizing cell metabolism with autofluorescence lifetime imaging
�Future prospects
Imagining imaging’s future
NATURE SS20 | SEPTEMBER 2003
Imagining imaging’s future
NATURE SS20 | SEPTEMBER 2003MEG: Magnetoencephalography AOH: Acoustic Optics Hybrid
Starting in the 16th century from a magnifier
The 19th century saw dramatic progress in the development of the microscope, thanks to the contributions of such great minds as Carl Zeiss, who devoted significant effort to the manufacture of microscopes, Ernst Abbe, who carried out a theoretical study of optical principles, and Otto Schott, who conducted research on optical glass.
Diffraction Limits
M. Minsky, US Patent 3013467, Dec. 19, 1961
T. Wilson and C.J.R. Sheppard, Theory and Practice of Scanning Optical Microscopy, (Academic, London, 1984)
Peter Török, P. Varga, Z. Laczik and G. R. Booker, Electromagnetic diffraction of light focused through a planar interface
between materials of mismatched refractive indices:
an integral representation, J. Opt. Soc. Am. A, 12, 325( 1995)
Confocal Microscopy
Marvin Minsky 1957
Confocal Microscopy
Z Koana 1943
Petrán 1968
16ms 20ms 24ms 28ms
Post-electrostimulation changes in Ca²+ in mouse ventricular cardiac muscle cell labeled with Fluo-3 , images taken at 4 ms intervals
Oxford microscope, 1975
Amar Choudhury, Colin Sheppard, Pete Hale and Rudi Kompfner
Oxford, Summer 1976
Marvin Minsky 1957
Confocal Microscopy
Z Koana 1943
One of the Microscopy Challenges:Elucidating Molecular Dynamics in Biology
without Destroying the MoleculesDiffract Limit: A barrier originated from the Uncertainty Principle
Difficult for life science to be alive at λ λ λ λ < 450 nm
Diffraction Limit: ∆∆∆∆R~λλλλ/NA
Ernst Abbe memorial at
Jena, Germany:
Nanoscopy:Seeing Beyond the Diffraction Limit
1. Single Fluorophore Localization microscopy
(a) Stochastic optical reconstruction microscopy
(STORM), 2006, Xiaowei Zhuang
(b) Photoactivated localization microscopy
(PALM), 2006, by Eric Betzig, Harald Hess, and
Samuel Hess.
(c) SPDM C. Cremer, 2008.
2. Saturated structured illumination microscopy
(SSIM) by Mats Gustafsson.
3. Stimulated emission depletion (STED) by Stefan
Hell.
From micro to nano
200 nm �100 nm � 30 nm (� 10 nm)
Optical Localization Microscopy
1. Single molecule (or fluorophore)
activation
2. Single molecule detection
3. Centralizing
4. Image reconstruction
Courtesy of Eric Betzig
A moiré pattern and structured illumination
Mats Gustaffsonhttp://www.bioengineering.ucsf.edu/faculty-
mats_gustafsson.vp.html
Courtesy of Mats Gustaffson
NATURE BIOTECHNOLOGY V21 # 11 NOVEMBER 2003
Courtesy of Stefan Hell
4Pi Microscopy
http://www.mpibpc.gwdg.de/abteilungen/200/4Pi.htmhttp://www.mpibpc.gwdg.de/abteilungen/200/mmm.html
http://www.uchsc.edu/lightmicroscopy/sted4pi.htm
Courtesy of Stefan Hell & Alex Egner
STED-4Pi Microscopy
http://www.mpibpc.gwdg.de/abteilungen/200/STED.htm
http://www.mpibpc.gwdg.de/groups/hell/
UV microscope
(Near field microscope)
Structured illumination
STED
PALM & STORM
Range of Molecular Interaction
The gap of optical microscopy
http://www.mindfully.org/Technology/2004/Buckyball-Football1jul04.jpg
Nanoscopy:
Seeing Beyond the Diffraction LimitFrom micro to nano
200 nm �100 nm � 30 nm (� 10 nm)
Limits:1. Note that the critical distance for bio-interaction is ~10 nm.
2. The photon statistics
3.The “brute force” dimension
the “free” dimension
PSF Volume ( )4
1
NA∝
The solution may lie in the temporal domain.
Starting with Molecular States __Franck-Condon and Jablonski Energy Diagram
Dipole-dipole interaction1. Van der Waal force
2. FRET (Förster/Fluorescence Resonant Energy Transfer)
2 23
κ ≈
p2A→→→→
p2B→→→→p2
C→→→→
E1→→→→
E1→→→→
E1→→→→r̂
θθθθ1111 →→→→2222
^
r→→→→
θ1
p2^
E1→→→→
⊥⊥⊥⊥∧∧∧∧
E1→→→→
p1→→→→
⊥⊥⊥⊥∧∧∧∧⊥⊥⊥⊥
∧∧∧∧ kT =1
τrD
R0
R
6
Criteria of FRET
• Overlapping of the emission spectrum of D and the absorption spectrum of A. (> 30%).
• Close proximity of two fluorophores (within 1 to 10 nm).
• Favorable mutual orientation.
• High quantum yield of the donor.
R
κ κ κ κ : orientation factor
D A
Fluorescence
photon
Laser pulseFluorescence
photon
Many idling cycles (for
avoiding piling up)
Start-stop-time 1 Start-stop-time 2
Histogram of correlated interval
Fluorescence Lifetime Measurement
1. The concept of a very fast stop watch2. Naturally compatible with confocal microscope using pulsed lasers.
A Time-domain Technique: Time Correlated Single Photon Counting (TCSPC)
2-p Time-correlated single photon counting microscopy
FLIM = Confocal+ Pulsed laser + Time-resolved module
The Advantages of Photon Counting
• High temporal resolution (~0.1 ns)
• Great signal-to-noise ratio (the SMD capacity)• Intensity fluctuations in light source is not critical• Intuitive data interpretation
The Significance of FLIM/FRET
• To distinguish “co-localization” and “binding” with nanometer (1~10 nm) sensitivity
• in vivo and in situ observation
FLIM is far more quantitative than ratio imaging (intensity FRET), not affected by photobleaching, dilution, ..etc.
Scope of FLIM/FRET:Detection of Bio-interactions and Micro-environment
changes that may be revealed by dipole-dipole interaction
1. Cell-to-Cell Signaling: ligand/receptor
2. Immune System: antigen-antibody interactions
3. Expression of Genes : DNA/regulator protein interactions
4. Metabolism: Formation of enzyme complex
5. Cell Division: DNA hybridization
6. Protein Folding
7. Changes of micro-environment surrounding the reporting fluorophores
Cell Vitality and Metabolism
through
Auto-fluorescence
Autofluorescence_first demonstrated in the 50s of the 20th century
1. No labeling
2. Generally ignored due to its weakness
3. Very weak signal: photon statistics is important
4. Short excitation wavelength: 2-p excitation is
recommended
5. Broad spectral distribution: spectrum shift does not
provide much useful information
6. Lifetime may exhibit interesting information
FluorophoreMain exc. peaks
(nm)
Main emission peaks
(nm)
Fluorescence lifetime
(ns)
Tryptophan 275 350 2.8, 1.5
Collagen 340, 270, 285 295, 395, 310 9.9, 5.0, 0.8
Elastin 460, 360, 425, 260 520, 410, 490, 410 6.7, 1.4, 7.8, 2.6, 0.5
NADH 350 460 0.6, 0.2, 2.5, 6
FAD, Flavins 450 535 ~4
FMN 440 520 4.7
β-carotene 520 9.6, 2.0, 0.3
Porphyrins 400 610, 675 9.6, 2.0, 0.3
Lipofuscin 340-395 540, 430-460 -
The The autofluorescenceautofluorescence
300 700500 1100900 15001300 2100 3000 nm
Visible light spectrum
2-photon excitation Most staining dyesSi or GaAs semiconductorSHG
Spectral range of a small frame Ti:Sapphire laser
SHG of Ti:Sa
Kr-Ar
568
Nd:YVO4
532
GaN
403
Ti:Sapphire
He-Ne
633
Light source
λλλλ
Applications
Ar ion
514
He-Ne
543
Ar ion
488
Wide bandgap material Small moleculesGas phase sample
2-p UVB excitation Aromatic amino acidsWide bandgap semiconductor
Bio-window Low IR absorption3-photon processes (THG,EFISH) Interfaces
SHG signal idler
2100 3000
idler1600 2100
OPO signal1350
1300
OPOsignal
1050650525
pump pump
920460 700350
Ar ion
365
Light Sources for Confocal Microscopy
Solid State Laser
The Promises of Ultrafast laser
Nonlinear Optics (SHG, THG, 2-p)Spectral and Time-resolved StudiesFrequency Comb (Nobel 2005)…..
Coming Opportunities on
Optical Microscopy
1. Stimulated Emission_ Stefan Hell & Sunny Xie
2. Fiber laser and Photonics Crystal Fiber_ Nonlinear
optics, Spectroscopy
3. Hybrid detectors__ high QE, large area, and fast
4. Polarization resolving
5. Photon Statistics_ Single Molecule Detection,
Physics at work
Retro-
Reflector
Retro-
Reflector
Long
pass
½ wave
plate
+Polarizer
½ wave
plate
800
NIR
50/50
ultrafast
beam splitter
½ wave
plate
+Polarizer800
NIR800
NIR
800
NIR
800
NIR
800
NIR
800
NIR
800
NIR
Long
passObjectiv
e
20X
Objectiv
e
40X
PCF
800 nm
RELP
FLIM CARS Fluorescence Lifetime Imaging
Live HuH (human hepatoma) liver cells
FLIM
Hoescht:
long
lifetime
DiO: short
lifetime
CARS
0 ns 1 ns 3 ns2 ns
Albert Stolow: This worked
extremely well and, within days, the
team was able to demonstrate - for
the first time - a new capability: a
FLIM-CARS microscope system…..
137mm
105mm
39mm
Mechanical Base Design Mechanical Base Design
50
22--Axis VCM and Pickup ModuleAxis VCM and Pickup Module
150mm
130mm
130mm
X-axis
Y-axis
Z-axis
AdjustStage
X-Y Stage for NRC Project::::Resolution of X axis: 0.5 um
Resolution of Y axis: 0.5 um
Thank You for Your Attention !