#### Transcript Document

```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
Ernst Abbe, 1873
d
1.22 
2n sin 
500 nm
 Methods that are based on
lenses have limited spatial
resolution
 Where does this result
originate?
200 nm
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Basic Concepts - Wave Optics
• Interference
• Diffraction
QuickTime™ and a
Animation decompressor
are needed to see this picture.
Christiaan Huygens – Treatise on Light
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Numerical Aperture & Resolution I
NA  nsin( )


The NA is one of the most important
parameters of an optical microscope.
2a
It determines:
• The amount of collected light
I  NA2
•The optical resolution
z
d  0.61
NA
•But where does it originate?
Note: tan  a
z
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Numerical Aperture & Resolution II
• What is the intensity distribution for a 2-D aperture  I(x, y, z)?
2
2a
2b

2
 kxa   
 kyb  
I(x, y, z)  E(x, y, z)  sinc 
  sinc 


z
z  

 
2
Note: sinc q sinq
q
Intensity on screen
• Sketching the result:
: 1b
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: 1a
Numerical Aperture & Resolution III
• Where are the zeroes of the intensity function?
 kxa 
sinc 
 z 
xˆ 
z
ka

z
2a
• Adding one final substitution & approximation:
tan  
a
z
for small 
 sin   tan  
• Gives:
xˆ 
z
2a


2 sin 

n•
2NA
• For air, n = 1:
square aperture round aperture
xˆ 
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0.5 
NA
xˆ 
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0.61
NA
a
z
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 
r [nm] with NA = 0.3,  = 500 nm
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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 
QuickTime™ and a
Animation decompressor
are needed to see this picture.
r [nm] with NA = 0.3,  = 500 nm
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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 
r [nm] with NA = 1,  = 500 nm
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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:
Magnification
NA
16x
20x
40x
63x
100x
0.45
0.7
1.3
1.4
1.3
.61
R
NA
Resolution [nm]
 = 400 nm
542
nm
349
nm
188
nm
174
nm
188
nm
Resolution [nm]
 = 600 nm
813
nm
523
nm
282
nm
261
nm
282
nm
r [nm] with NA = 0.3,  = 500 nm
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Practice: High-resolution optical methods
~250 nm
~180 nm
~30 nm
~100 nm
~30 nm
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Garini et al, Curr Opin11Biotech 2005. 16, 3-12
How can we overcome the diffraction limit??
Completely different
approach:
NEAR FIELD
~50 nm
High intensity
Low intensity
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):
Different schemes:
single/multi layers
high/low voltage
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Near-field microscope:
feedback mechanism
Tuning fork
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Near-field scanning optical microscope
(NSOM or SNOM)
The tip must be ~10 nm from the sample
psd
laser
optical fiber
laser
Piezo 3-axis motor
tip
sample
collection optics
detector
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Tip – atom interaction:
Van der Waals potential
Potential
Energy
r
Repulsion
Attraction
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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
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Near-field
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Total Internal Reflection Microscopy
Principle: Happens when light hit a surface
θ>θc & n1>n2
Calculation of θc for n1=1.5, n2=1.36 → Use Snell’s law:
n1 sin 1  n2 sin  2
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n 
 1  arcsin  2   65 0
 n1 
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Total Internal Reflection Field
creates evanescent field
z
I z  I 0 ez d
d
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
4 n12 sin 2 1  n22
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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
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Virometer: An Optical Instrument for Visual Observation, Measurement and
Classification of Free Viruses, Hirschfeld T, Block M, Mueller W, J. Histochemistry &
Cytochemistry, 25:719-723 (1977).
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TIRF History II
• What can we measure in this thin excitation field?
electron microscope diameter
• Dynamic movement of labeled biomolecules
Protein dynamics
Vesicle–actin dynamics
virometer Brownian diameter
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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: TIRF
Gregg Gundersen, Columbia University
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Advanced TIRF for single molecule detection
Setup:
Interference
Calibration by moving
the slide
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Cappello, G. Physical Review E 68, 2003.
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Hyper-spectral microscopy
Garini (1996):
Using chromosome-specific probes & markers
EXCITATION and EMISSION SPECTRA
Cy2
SpectrumGreen
FITC
Cy3
Rhodamine
SpectrumOrange
Texas Red
Cy3.5
Cy5
Cy5.5
DAPI
INTENSITY [arb. units]
EXCITATION
EMISSION
400
450
500
550
600
650
WAVELENGTH [nm]
700
750
800
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,
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BarAm I, Soenksen D, Garini Y, Ried T, Science 273:494-497 (1996).
Hyper-spectral microscopy II
Sagnac interferometer
CCD detector
collimating lens
light source
filter cube
objective
sample
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• For every pixel (x,y) on
the CCD camera a
complete spectrum is
generated
• This permits
classification on the
basis of color
Hyper-spectral microscopy III
• This, in turn, permits spectral karyotyping
And the detection of
genetic abnormalities…
And
recognition…
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FLIM
Arndt-Jovin (1979):
4
3
2
n=1
Dt ≈ 10 ns 
excited electrons [%]
100%
• 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
80%
60%
Bodipy TR  = 4.85 ns
Nile Red
 = 2.71 ns
40%
20%
0%
0
2
4
6
8
10
time [ns]
• Fluorescence Decay Analysis in Solution and in a Microscope of DNA and
Chromosomes Stained with Quinacrine, Arndt-Jovin DJ, Latt S, Striker SA, Jovin
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TM, J. Histochemistry & Cytochemistry, 27:87-95 (1979).
FLIM
• There are several ways to measure this phenomenon:
• Sinusoidal light source modulation (now with LEDs!)
• Pulse method
• Gated method
• PRBS light source modulation
Steady-state intensity image Time-resolved intensity image
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