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STED: Nanoscale 3D Optical Imaging
Digvijay Raorane & Arun Majumdar
Department of Mechanical Engineering
Department of Materials Science
University of California, Berkeley
Materials Sciences Division
Lawrence Berkeley National Laboratory
Outline
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Motivation
Introduction: Conventional Optics
Near Field Imaging
STED
- Theory
- Previous experimental work
• On-going experiments
Biological Imaging
•
Virus
TMV 16.5 nm
•
Microtubules
Microtubule 25 nm
• Cell Organelle
ER canaliculi dia. 40-60 nm
Need for High Resolution Optical
Technique
• Biomolecules that require imaging are typically 1- 50 nm in
size
• Far-field optics (e.g. confocal) limited to resolution > 200
nm, which cannot directly resolve molecular-scale phenomena
• Atomic Force Microscopy cannot be used inside a cell
• Optical/fluorescence imaging is most-widely used
approach for real-time intracellular visualization
• NSOM (Near-field Scanning Optical Microscope)
Optical Imaging at a Glance
Far-Field Optics
Near-Field Scanning
Optical Microscope (NSOM)
d
Diffraction-limited spatial
resolution ~ λ/2*NA
Aperture-limited spatial
resolution d ~ 50 nm
NSOM Limitations
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Single fiber is limited to the field of view to ~ 50
nm.
•
It is difficult to maintain the tip at the constant
distance from the sample within few nms.
•
Tip can get damaged by the thermal stress due
to the light.
•
Scanning a whole cell area (10 mm x 10 mm)
takes time.
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Multi-location imaging and dynamics cannot be
observed.
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Fiber-drawing and aperture fabrication is not
repeatable, producing different imaging
conditions each time.
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Tip may get clogged when biological sample is
in its buffer medium.
Tip Profile
http://micro.magnet.fsu.edu
Tip Damage
Rosa et al., Appl. Phys. Lett. 67,
(18), 2597-2599 (1995)
What is Stimulated Emission
Depletion (STED) Microscopy?
Spontaneous Emission Fluorescence
Absorption
Spontaneous Vibrational
ex
Relaxation
Emission
Absorption
 ~10 ns
Emission
ex
Vibrational
Relaxation
Wavelength, 
sp-em
Fluorescence Imaging
Diffraction-limited spatial
resolution ~ λ/2*NA
Stimulated Emission
Absorption
ex
st
Vibrational
Relaxation
Absorption
st
st
Emission
ex
Vibrational
Relaxation
Wavelength, 
st
Physical Realization
• Conceptual Set-up
STED Spot
Avalanche
Photodiod
e
λ/2 Phase
Plate
Fluorescence
645-715 nm
Excitation Spot
Fluorescence
Imaging
Spot, d<< /2*(NA)
Hell S. et al.,Nature Biotech., 21(11), 2003
Point Spread Function Engineering
• Non Linear Optical Effect
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STED laser quenches tail of PSF due to excitation laser => reduction in
FWHM of resultant focal spot incident on fluorescence sample
Weiss S. et al., PNAS, 97 (16),pg. 8747–8749,2000
Resolution
• Resolution (FWHM) dependence on Intensity
8

r 

max
2n  Sin 
I STED
 1
I saturation
FWHM (Δr)
• For typical experiment,
MW
max
  700nm, I STED  800 2 ,
cm
max
I STED
 36, n  151
. ,   680
I sat
Hence, r  37nm
STED Imaging
Absorption
ex
img st
Vibrational
Relaxation
Absorption
st
st
Emission
ex
Vibrational
Relaxation
Wavelength, 
st
Proof of Concept
Al2O3 matrix wetted by Polymethyl
Methacrylate
Westphal et al, APL, 82(18), 3125 - 3127 (2003)
Westphal et al,PRL 94, 143903 (2005)
Experimental Set up
Ti:Sa
Laser
st
OPO
Delay
LPC
PS
ex
CH
st
SAMPLE
Fluorescenceimg
Detector
DC 1
DC 2
Collaboration: Prof. Costas Grigoropoulos (ME Dept, UCB)
On-Going Work
• Quantum dot as substitute to fluorescent tags
- To test compatibility of Q Dots with STED microscopy to overcome
photobleaching of fluorescent labels
d (nm)
CdSe
Nucleus
with actin
fibres
Hines et al,Advanced Materials,15, 1845, 2003
Alivisatos et al, Nature Biotech., 22, 47 – 52, 2004
d
4.6
3.6
3.1
2.4
2.1
Advantages of STED Microscopy
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High resolution can be achieved
routinely ( < 50 nm)
No need of probe/tip
Signal can be collected at far-field
Better than confocal microscope in
terms of resolution
Multiple areas can be probed by
forming multiple spots on the sample
Resolution depends on the laser
intensity ( FWHM = f( Intensity))
Incorporates most widely used
fluorescence technique by
biologists
Can image live biological sample
Optical system is simple to
understand
Can scan the sample in z direction
for 3 D image
3 D Image
Yeast
Mammalian
Mitochondria Golgi
Hell et al, J. Opt. Soc. Am. A , 9(12),
2159 – 2166, 1992