STED: Nanoscale 3D Optical Imaging

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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
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Outline

• Motivation
• Introduction Conventional Optics
• Near Field Imaging
• STED
• Theory
• Previous experimental work
• On-going experiments

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Biological Imaging

• Virus
• Microtubules
• Cell Organelle

TMV 16.5 nm
Microtubule 25 nm
ER canaliculi dia. 40-60 nm
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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 gt 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)

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Optical Imaging at a Glance
Near-Field Scanning Optical Microscope (NSOM)
Far-Field Optics
Aperture-limited spatial resolution d 50 nm
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NSOM Limitations
Tip Profile

• 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.
• Multi-location imaging and dynamics cannot be
  observed.
• Fiber-drawing and aperture fabrication is not
  repeatable, producing different imaging
  conditions each time.
• Tip may get clogged when biological sample is in
  its buffer medium.

http//micro.magnet.fsu.edu
Tip Damage
Rosa et al., Appl. Phys. Lett. 67, (18),
2597-2599 (1995)
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What is Stimulated Emission Depletion (STED)
Microscopy?
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Spontaneous Emission - Fluorescence
Absorption
Vibrational Relaxation
? 10 ns
Absorption
Emission
Vibrational Relaxation
Wavelength, ?
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Fluorescence Imaging
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Stimulated Emission
Absorption
Vibrational Relaxation
?ex
Absorption
Emission
?ex
Vibrational Relaxation
Wavelength, ?
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Physical Realization

• Conceptual Set-up

Excitation Spot
Hell S. et al.,Nature Biotech., 21(11), 2003
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Point Spread Function Engineering

• Non Linear Optical Effect
• STED laser quenches tail of PSF due to
  excitation laser gt reduction in
• FWHM of resultant focal spot incident on
  fluorescence sample

Weiss S. et al., PNAS, 97 (16),pg. 87478749,2000
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Resolution

• Resolution (FWHM) dependence on Intensity
• For typical experiment,

FWHM (?r)
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STED Imaging
Absorption
Vibrational Relaxation
?ex
?st
?st
Absorption
Emission
?ex
?st
Vibrational Relaxation
Wavelength, ?
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Proof of Concept
Al2O3 matrix wetted by Polymethyl Methacrylate
Westphal et al,PRL 94, 143903 (2005)
Westphal et al, APL, 82(18), 3125 - 3127 (2003)
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Experimental Set up
Delay
?st
TiSa Laser
LPC
OPO
PS
?ex
CH
?st
SAMPLE
?img
Fluorescence
Detector
DC 1
DC 2
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Collaboration Prof. Costas Grigoropoulos (ME
Dept, UCB)
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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

Nucleus with actin fibres
Hines et al,Advanced Materials,15, 1845, 2003
Alivisatos et al, Nature Biotech., 22, 47 52,
2004
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Advantages of STED Microscopy
3 D Image

• High resolution can be achieved routinely ( lt 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

Yeast Mitochondria
Mammalian Golgi
Hell et al, J. Opt. Soc. Am. A , 9(12), 2159
2166, 1992