ECE212CN: Nano-Photonics Lecture 10 High Resolution...
Transcript of ECE212CN: Nano-Photonics Lecture 10 High Resolution...
ECE212CN: Nano-Photonics
Lecture 10
High Resolution Optical Imaging Techniques
Near-field Scanning Optical Microscopy (NSOM) and
Stimulated emission depletion microscopy (STED)
Near-field Scanning Optical MicroscopyNear-field scanning optical microscopy: NSOM Scanning near-field optical microscopy: SNOM
A Short History of NSOM/SNOM
1928/1932 E.H. Synge proposes the idea of using a small aperture to image a surface with sub-wavelength resolution using optical light. [E.H. Synge, "A suggested method for extending the microscopic resolution into the ultramicroscopic region" Phil. Mag. 6, 356 (1928); E.H. Synge, "An application of piezoelectricity to microscopy", Phil. Mag., 13, 297 (1932)]. The proposal, although visionary and simple in concept, was far beyond the technical capabilities of the time.
1956 J.A. O'Keefe, a mathematician, proposes the concept of Near-Field Microscopy without knowing about Synge's earlier papers. However, he recognizes the practical difficulties of near field microscopy and writes the following about his proposal: "The realization of this proposal is rather remote, because of the difficulty providing for relative motion between the pinhole and the object, when the object must be brought so close to the pinhole." [J.A. O'Keefe, "Resolving power of visible light", J. of the Opt. Soc. of America, 46, 359 (1956)]. In the same year, Baez performs an experiment that acoustically demonstrates the principle of near field imaging. At a frequency of 2.4 kHz (14cm), he shows that an object (his finger) smaller than the wavelength of the sound can be resolved.
1972 E.A. Ash and G. Nichols demonstrate λ/60 resolution in a scanning near field microwave microscope using 3 cm radiation. [E.A. Ash and G. Nichols, "Super-resolution aperture scanning microscope", Nature 237, 510 (1972)].
1984 The first papers on the application of NSOM/SNOM appear. These papers are the first to show that NSOM/SNOM is a practical possibility, spurring the growth of this new scientific field.
A. Lewis, M. Isaacson, A. Harootunian and A. Murray, Ultramicroscopy 13, 227 (1984);
B. D.W. Pohl, W. Denk and M. Lanz, APL 44, 651 (1984).
0.5 mm lines and 0.5 mm gaps
25nm size can be recognized using 488nm light. λ/20
Science 1991, 251, 1468-1470
Commercialized Products
Basic Principle
Light in tiny holesBethe, H. A. Theory of diffraction by small holes. Phys. Rev. 66, 163–182 (1944).
Diffraction and typical transmission spectrum of visible light through a subwavelength hole in an infinitely thin perfect metal film
A cylindrical waveguide with a radius r much smaller than the wavelength λ of the incident EM field milled in a metal film of thickness h.
Nature, 445, 39, (2007)
Light in tiny holesNature, 445, 39, (2007)
Optical transmission properties of single holes in metal films.The holes were milled in suspended optically thick Ag films illuminated with white light. a, A circular aperture and b, its transmission spectrum for a 270nm diameter in a 200-nm-thick film. c, A rectangular aperture and d, its transmission spectrum as a function of the polarization angle h for the following geometrical parameters: 210nm3310 nm, film thickness 700 nm.
Near field scanning OM (NSOM)
Tip + Scanner and feedback controller + Detection
Tips
Static and dynamic chemical etching
Laser pulling
AFM tip based
Tips
Metal Coating Focusing Ion Beam Milling
Polarization dependent
Tips (Apertureless)
Metalic tip
E.J. Sanchez et al., Phys. Rev. Lett. 82, 4014 (1999).L. Novotny et al., Ultramicroscopy 71, 21 (1998).
Resolution Polarization Others
Apertured In-plane Less dose
Apertureless Higher Vertical
Scanner and feedback controller
Oscillatory Feedback Methods Tapping-Mode Feedback
Detection
Phys. Rev. Lett. 85, 3029 (2000)
Complex field measurement Pseudoheterodyne detection for background-free near-field spectroscopy
Appl. Phys. Lett. 89, 101124 (2006)
Examples
Surface plasmons on a metal surface
Stimulated emission depletion microscopy
Stefan Hell
Theoretical paper
ni: population probabilitiesQ: quenching rateσ: transition cross sectionh: intensity distribution
Stimulated emission depletion
Two pulse beam: 1. Molecules excitation beam (diffraction
limited spot)2. Depletion beam (‘STED-pulse’)
The net effect of the STED pulse is that the affected excited molecules cannot fluoresce because their energy is dumped and lost in the STED pulse.
Spatial arrangement of the STED pulse and excitation pulse
STED-pulse size:Diffraction limited
Why the effective point spread function smaller than diffraction
limit?
Saturation of the fluorescence reduction
Isaturation tens to a hundred of MW/cm2
[1]. http://www3.mpibpc.mpg.de/groups/hell/; [2]. http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html
Experimental demonstration
ConfocalPSF
STEDPSF
Axial PSF
Lateral resolution: 2x improvementFWHM of 104 nm and 127 nm in x and y
directions, respectively
Axial resolution: 5x improvementFWHM of 97nm
[3]. Thomas A. Klar et. al., PNAS, vol. 97, no. 15, (2000)
Excitation beam
STED beam
Synchronized pulsed lasers
π
phase=0
phase plate for STED beamLocation: back aperture of the
objective lens
Conventional
STEDSTED
+deconvolution
Further lateral resolution improvement
[4]. V. westphal et. al., Appl. Phys. B 77, 377–380 (2003)
0π
phase plate for STED beamSynchronized pulsed lasers
(c) The idealized phase-only pupil functions with the optimal values of the
parameters d & h(d) sections of the
correspondinginhibition patterns are
Various STED beam
[5]. Jan Keller et. al., OPTICS EXPRESS, Vol. 15, No. 6, (2007)
3D XY X Y 3D XY
circular polarized
Resolution improvement in
STED beam profile relates to
(1)2D or 3D improvement
(2)Resolution improvement ratio
Most frequently used phase plate
[6]. Katrin I. Willig, Nature, Vol 440, 935, (2006)
Is: Threshold intensity for depletionIm: STED beam doughnut crest intensity
typically 0.1–1GW/cm2
Circular polarized light
Synchronized pulsed lasers
Imaging application
[7]. Gerald Donnert et. al, PNAS, vol. 103, no. 31, 2006
Resolve sub-diffraction scale features
Resolving the nanostructure of speckles of protein SC35 in intact
mammalian cell nuclei. (a, c, and e)
LD: linear deconvolution
Conventional
Imaging application
[8]. Gerald Donnert et. al, PNAS, vol. 103, no. 31, 2006Imaging neurofilaments in human neuroblastoma
Imaging application
Mechanism of synaptic labelling Labeled neuron
Reveal that
synaptotagmin remains
clustered after synaptic
vesicle exocytosis
Synaptic vesicles are too
small (~40nm in
diameter) and too
densely packed.
[9]. Katrin I. Willig, Nature, Vol 440, 935, (2006)
Recent progress - Video-Rate STEDHigh speed modification:
(1). Use a 16kHz resonant mirror to scan the excitation and depletion beams along 1 axis
(2). Use a piezo-actuator to scan the sample along the perpendicular axis
28 fps, spot size: 62nm, Field of view: 2.5x1.8 μm2
Frame rate depends on field of view
Circular polarized
light
Recent progress - Video-Rate STED
Video-Rate STED
[11]. Volker Westphal et. al, Science, vol 320, (2008)Characteristics of synaptic vesicle movement.
Further improve z resolution: 4pi – STED
[12]. Marcus Dyba et. al, Physical Review Letters, VOLUME 88, NUMBER 16, (2002)
Theoretical Excitation
Theoretical STED
Theoretical effective
PSF
Theoretical effective
PSF
Further improve z resolution: 4pi – STEDExperimental results
Oil immersion objective
Water immersion objective
Images of membrane-
labeled bacteria
Confocal
STED-4pi
STED-4pi deconv.
STED with continuous wave beamsPrevious STED microscopy:
Tightly synchronized trains of pulses
Typically excitation pulses of ~80 ps duration
followed by 250ps pulses for STED
Matching STED beam wavelength to the
emission spectrum of the dye:
Tunable pulsed lasers (such as Ti:sapphire laser
with frequency doubler)
Strech its ~250fs pulse by 1,000 fold using
optical fibers
About 4x larger
[13]. Katrin I Willig et. al, Nature Method, VOL.4 NO.11, (2007)
If dye satisfy kSTED > kfl > kexc, k is the
decay rate, STED concept works with
CW-STED beam
• CW-STED beam power 3~5 times
larger than the time-averaged power
of the pulsed system
• CW-STED beam intensity 10-15
fold weaker than peak intensity of the
pulsed system
To simplify instrument è CW-STED
STED with continuous wave beams
Low-power STED by time gating
[14]. Giuseppe Vicidomini et. al., Nature method, 2011.
Conventional STED microscopy:Temporal separation between excitation and de-
excitation, and the low time-averaged powerTightly synchronized trains of pulses
Typically excitation pulses of ~80 ps duration followed by 250ps pulses for STED
Matching STED beam wavelength to dye emission spectrum: Tunable pulsed lasers (such as Ti:sapphire
laser with frequency doubler) with pulse strecher
If dye satisfy kSTED > kfl > kexc, k is the decay rate, STED concept works with CW-STED beam
• CW-STED beam intensity 10-15 fold weaker than peak intensity of the pulsed system
èless prone to inducing multiphoton processes known to stress the sample
2011, Nature method, Stefan Hell group
fluorescence from a single isolated nitrogen vacancy (NV)
color centers in diamond
Tg=15 ns
To reach same level of depletion, CW-STED beam power 3~5 times larger than the time-averaged power of the pulsed system
If using same time averaged power è Less resolution improvement
Low-power STED by time gating