Cyttron NSOM Lecture

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Cyttron NSOM Lecture A Surface Imaging
Method Prof. Ian T. Young Quantitative Imaging
Group Department of Imaging Science
Technology Delft University of Technology
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Theory Optical resolution is limited

• Methods that are based on lenses have limited
  spatial resolution
• Where does this result originate?

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Basic Concepts - Wave Optics

• Interference
• Diffraction

Christiaan Huygens Treatise on Light
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Numerical Aperture Resolution I

• The NA is one of the most important parameters of
  an optical microscope.
• It determines
• The amount of collected light
• The optical resolution
• But where does it originate?

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Numerical Aperture Resolution II

• What is the intensity distribution for a 2-D
  aperture ? I(x, y, z)?

• Sketching the result

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Numerical Aperture Resolution III

• Where are the zeroes of the intensity function?
• Adding one final substitution approximation
• Gives
• For air, n 1

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Numerical Aperture Resolution IV

• A point of light as object produces an Airy disk
  as the 2-D image
• Two points of light produce two Airy disks
• The size of the Airy disk(s) depends on the NA
  and ?

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Numerical Aperture Resolution IV

• A point of light as object produces an Airy disk
  as the 2-D image
• Two points of light produce two Airy disks
• The size of the Airy disk(s) depends on the NA
  and ?

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Numerical Aperture Resolution V

• A point of light as object produces an Airy disk
  as the 2-D image
• Two points of light produce two Airy disks
• The size of the Airy disk(s) depends on the NA
  and ?

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Typical Values

• A round aperture produces an Airy disk on the
  screen
• The size of the Airy disk(s) depends on the NA
  and ?
• Rayleigh criterion says resolution is

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Practice High-resolution optical methods
250 nm
180 nm
30 nm
100 nm
30 nm
Garini et al, Curr Opin Biotech 2005. 16, 3-12
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How can we overcome the diffraction limit??
Completely different approach NEAR FIELD
Measure VERY CLOSE to tip 10 nm
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Example of a near-field tip
50 nm hole
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Near Field Microscopy
But how does it work? It can only detect one
small point. ? Need to scan the surface ? need
scanning mechanism with 10 nm resolution It uses
piezoelectric elements (expand with voltage)
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Piezoelectric motors
Material (example) Perovskite-type lead
zirconate titanate (PZT). Different
schemes single/multi layers high/low voltage
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Near-field microscope feedback mechanism
Tuning fork Optical probe
quadrant detector
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Near-field scanning optical microscope(NSOM or
SNOM)
The tip must be 10 nm from the sample
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Tip atom interactionVan der Waals potential
Potential Energy
r
Repulsion
Attraction
r
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NSOM working modes
Non-contact mode
Contact mode
Tapping mode
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NSOM example of a Muscle Tissue
Topography
Near-field
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Total Internal Reflection Microscopy
Principle Happens when light hit a surface ?gt?c
n1gtn2 Calculation of ?c for n11.5, n21.36 ?
Use Snells law
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Total Internal Reflection Fieldcreates
evanescent field
z
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Why is TIRF interesting?
Provides high resolution along z overcomes
wide-field limit
Limitation only measures the surface, Still
important for various applications.
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TIRF microscopy
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TIRF History

• Hirschfeld (1977)
• When light is reflected from a perfect mirror, a
  small amount of light (the evanescent wave) goes
  through to the other side of the mirror.
• The thickness of the wave on the other side is
  about ?/20, e.g. 25 nm.

Virometer An Optical Instrument for Visual
Observation, Measurement and Classification of
Free Viruses, Hirschfeld T, Block M, Mueller W,
J. Histochemistry Cytochemistry, 25719-723
(1977).
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TIRF History II

• What can we measure in this thin excitation
  field?
• Dynamic movement of labeled biomolecules

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TIRF examples
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TIRF examples
Cells labeled (tubulin) imaged with wide-field
(Center panel) and TIRF illumination.
Right Overlay of images. Green wide field, red
TIRFGregg Gundersen, Columbia University
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Advanced TIRF for single molecule detection
Setup Interference Calibration by
moving the slide
Cappello, G. Physical Review E 68, 2003.
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Advanced TIRF Results
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Hyper-spectral microscopy
Garini (1996) Using chromosome-specific
probes markers
DAPI
5 dyes are sufficient for 24 chromosomes
Multicolor spectral karyotyping of human
chromosomes, Schrock E, duManoir S, Veldman T,
Schoell B, Wienberg J, FergusonSmith MA, Ning Y,
Ledbetter DH, BarAm I, Soenksen D, Garini Y, Ried
T, Science 273494-497 (1996).
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Hyper-spectral microscopy II
For every pixel (x,y) on the CCD camera a
complete spectrum is generated This permits
classification on the basis of color
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Hyper-spectral microscopy III

• This, in turn, permits spectral karyotyping

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FLIM
Arndt-Jovin (1979)
There is a distribution of times associated
with the return of an electron to the ground
state and the emission of a photon The
biochemical environment (e.g. pH, O2, Ca2) of
the fluorescent molecule can affect this
fluorescence lifetime
Dt 10 ns ? fluorescence lifetime
Fluorescence Decay Analysis in Solution and in
a Microscope of DNA and Chromosomes Stained with
Quinacrine, Arndt-Jovin DJ, Latt S, Striker SA,
Jovin TM, J. Histochemistry Cytochemistry,
2787-95 (1979).
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FLIM

• There are several ways to measure this
  phenomenon
• Sinusoidal light source modulation (now with
  LEDs!)
• Pulse method
• Gated method
• PRBS light source modulation