Phys 570V: Advanced Topics in Optics and Photonics
Professor Tongcang Li
Lecture 24: Optical imaging beyond diffraction limit
Course website: http://www.physics.purdue.edu/academicprograms/courses/course_detail.php?SEM=fall2015&c=phys570V
Syllabus, Lecture notes, etc.
Purdue University
Fall 2015
Physics 570V
Room: Phys 331 Time: MW 2:30-3:45 PM
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Generation of entangled photon pairs
•
Cascade in atomic transitions (eg. Ca)
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Generation of entangled photon pairs
•
Down-conversion in nonlinear crystal
𝑘=
𝑛𝜔
𝑐
Phase matching
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This lecture: Optical imaging beyond diffraction limit
The Nobel Prize in Chemistry 2014
Eric Betzig, Stefan W. Hell, William E. Moerner
“for the development of super-resolved fluorescence microscopy"
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Abbe’s Diffraction limit
Abbe Resolutionx,y = λ/2NA
Abbe Resolutionz = 2λ/NA2
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Fluorescence microscopy
https://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html
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Content
• 1. confocal microscope
• 2. Near field scanning optical microscope (NSOM)
• 3. Two-photon optical microscope
• 4. Stimulated Emission Depletion Microscope (STED)
• 5. Single-molecule localization microscopes: PALM, STORM, etc.
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Confocal microscopy
A pinhole in the back focal plane rejects the light coming from outside the focal plane. The
pinhole size is a trade-off between good rejecting ability and sufficient light throughput
(typically ~ 30 – 150 mm)
wide field
CCD
PMT
MPD
…
dichroic
whole image at once
confocal
image is scanned point by point
Resolution in confocal microscopy:
Slightly higher resolution than in wide field microscopy (improvement ~ 1.4) when
a very small pinhole is used.
~ 3D Gaussian profile
The image is a convolution of the object and the PSF
Confocal vs. Wide field microscopy:
Wide-field:
Confocal:
Elimination of out-of-focus light improves contrast and, thus, resolution
Confocal microscopy:
Focusing only in one plane  axial sectioning of the sample to ~ mm slices
Near field scanning optical microscope (NSOM)
Journal of Applied Physics 59, 3318 (1986);
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Bell Jar
Hallen lab, NC State
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Signal Strength vs Resolution
Resolution only depends on aperture, not wavelength
Theoretical: 1/r6 scaling
50 nm practical limit
Hallen lab, NC State
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Scanning Probe Feedback Mechanism:
AFM and NSOM same implementation
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Near Field Scanning Optical
Microscopy/Spectroscopy (NSOM) of
Advanced Organic Thin Film Materials
Joseph Kerimo, David M. Adams,
David A. Vanden Bout, Daniel A.
Higgins and Paul F. Barbara
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Apertureless NSOM
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Limitations
• Shallow depth of view.
• Weak signal
• Difficult to work on cells, or other soft samples
• Complex contrast mechanism
• Slow
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Two-photon optical microscope
excited state
excitatio
n
emission
emission
excitatio
n
excitatio
n
ground state
One-photon excitation
Two-photon excitation
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The difference between single- and two-photon excitation
Optical sectioning
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Wide-field vs. confocal vs. 2-photon
Drawing by P.
D. Andrews, I.
S. Harper and
J. R. Swedlow
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Resolution of 2-photon systems
Using high NA pseudoparaxial approximations1 to estimate the illumination,
the intensity profile in a 2-photon system, the lateral (r) and axial (z) full
widths at half-maximum of the two-photon excitation spot can be
approximated by2:
 0.32  
NA  0.7
 2  NA
r0  
 0.325  
NA  0.7
 2  NA0.91
1)
2)
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
0.532 
1
z0 


2  n  n 2  NA2 
V2h   r z
32 2
0 0
C. J. R. Sheppard and H. J. Matthews, “Imaging in a high-aperture optical systems,” J. Opt. Soc. Am. A 4, 1354- (1987)
W.R. Zipfel, R.M. Williams, and W.W. Webb “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369-1377 (2003)
Practical resolution
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Centonze VE, White JG. Multiphoton excitation provides optical sections from
deeper within scattering specimens than confocal imaging. Biophys J. 1998
Oct;75(4):2015-24.
Effect of increased incident power on
generation of signal. Samples of acidfucsin-stained monkey kidney were
imaged at a depth of 60 µm into the
sample by confocal (550 µW of 532-nm
light) and by multiphoton (12 mW of
1047-nm light) microscopy. Laser
intensities were adjusted to produce the
same mean number of photons per pixel.
The confocal image exhibits a
significantly narrower spread of pixel
intensities compared to the multiphoton
image indicating a lower signal to
background ratio. Multiphoton imaging
therefore provides a high-contrast
image even at significant depths within
a light-scattering sample. Images were
collected at a pixel resolution of 0.27 µm
with a Kalman 3 collection filter. Scale
bar, 20 µm.
Bartek Rajwa
Stimulated Emission Depletion (STED) Microscope
Drive down to ground state with second “dump”pulse,
Before molecule can fluoresce
Quench fluorescence and Combine with spatial control
to make “donut”, achieve super-resolution in 3D (unlike NSOM)
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Setup
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Excitation and deexcitation beams for 3D STED
Hein B et al. PNAS 2008;105:14271-14276
Klar T A et al. PNAS 2000;97:8206-8210
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Resolution improvement in STED
Klar T A et al. PNAS 2000;97:8206-8210
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Example: Subdiffraction resolution fluorescence imaging of microtubules
Hein B et al. PNAS 2008;105:14271-14276
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Single-molecule localization methods: PALM, STORM, etc.
PALM: photoactivated localization microscopy
STORM: stochastic optical reconstruction microscopy
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Photo-active GFP
G. H. Patterson et al., Science 297, 1873 -1877 (2002)
This paper reported a
photoactivatable
variant of GFP that,
after intense
irradiation with 413nanometer light,
increases
fluorescence 100
times when excited
by 488-nanometer
light and remains
stable for days under
aerobic conditions
Native= filled circle
Photoactivated= Open squares
Wild-type GFP
T203H GFP:
PA-GFP
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Photoactivation and imaging in vitro.
G. H. Patterson et al., Science 297, 1873 -1877 (2002)
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https://www.microscopyu.com/articles/superresolution/stormintro.html
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Stochastic optical reconstruction microscopy (STORM)
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3-D (z) resolution
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Fig. 2. Three-dimensional STORM imaging of microtubules in a cell.
Conventional indirect
immunofluorescence
image of microtubules
3D section
(color coded)
C-E zoom in
of box in B
B Huang et al. Science 2008;319:810-813
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Final presentations: 11/30-12/9
Date
Presenter
11/30
Eric Topel
Mikhail
Shalaginov
Cong Wang
Robert
Sutherland
12/2
Dewan Woods
Jaehoon Bang
Yu Gong
Zhuoxian Wang
12/7
Nirajan Mandal
Di Wang
Jonghoon Ahn
Jin Cui
12/9
Ting-wei Hsu
Jie Hui
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Final presentations (12-minute talk + 3 minute Q&A).
Grading guide:
1. Content (technical comprehension and explanation of the topic)
2. Organization and visual aids (Logical sequence, text, graphics)
3. Verbal presentation (speaking volume, rate, eye contact, body language,
enthusiasm for the topic)
4. Overall impression (interesting talk, understanding and answering
questions correctly, etc.)
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