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