Cyttron 1 Cyttron NSOM Lecture A Surface Imaging Method Prof. Ian T. Young Quantitative Imaging Group Department of Imaging Science & Technology Delft.

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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

Ernst Abbe, 1873

500 nm

200 nm

Methods that are based on lenses have limited spatial resolution

Where does this result originate?

d =1.22λ2nsinθ

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Christiaan Huygens – Treatise on Light

Basic Concepts - Wave Optics

•Interference

•Diffraction QuickTime™ and aAnimation decompressor

are needed to see this picture.

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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?

NA=nsin(θ)

I ∝NA2

d≈0.61λNA

2aθ

θ

z

Note:  tanθ =az

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

•Sketching the result:

I (x, y, z) = E(x,y,z) 2 = sinckxaz

⎛⎝⎜

⎞⎠⎟

⎡

⎣⎢⎤

⎦⎥

2

  sinckybz

⎛⎝⎜

⎞⎠⎟

⎡

⎣⎢⎤

⎦⎥

22a

2b

Intensity on screen

 :   1

b

 :   1

a

Note: sinc q( )=sinqq

Numerical Aperture & Resolution II

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•Where are the zeroes of the intensity function?

•Adding one final substitution & approximation:

•Gives:

•For air, n = 1:

tanθ =az

for small θ ⇒ sinθ ≈tanθ =az

x̂ =λz2a

=λ

2sinθ=

n• λ2NA

square aperture round aperture

x̂ =0.5λNA

x̂=0.61λNA

x̂ =πzka

=λz2a

sinckxa

z⎛⎝⎜

⎞⎠⎟

Numerical Aperture & Resolution III

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•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 λ

r [nm] with NA = 0.3, λ = 500 nm

Numerical Aperture & Resolution IV

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•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 λ

r [nm] with NA = 0.3, λ = 500 nm

Numerical Aperture & Resolution IV

QuickTime™ and aAnimation decompressor

are needed to see this picture.

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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 λ

r [nm] with NA = 1, λ = 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 λ

•Rayleigh criterion says resolution is:

R=.61λNA

Magnification NA

16x 0.45 542 nm 813 nm20x 0.7 349 nm 523 nm40x 1.3 188 nm 282 nm63x 1.4 174 nm 261 nm100x 1.3 188 nm 282 nm

Resolution [nm]λ = 400 nm

 [ ]Resolution nmλ = 600 nm

r [nm] with NA = 0.3, λ = 500 nm

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~250 nm

~180 nm

~100 nm

~30 nm

~30 nm

Garini et al, Curr Opin Biotech 2005. 16, 3-12

Practice: High-resolution optical methods

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How can we overcome the diffraction limit??

Completely different approach:

NEAR FIELD

High intensity

Low intensity

~50 nm

Measure VERY CLOSE to tip ~10 nm

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50 nm hole

Example of a near-field tip

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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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tip

laser

collection optics

detector

sample

optical fiber

psdlaser

Piezo 3-axis motor

Near-field scanning optical microscope

(NSOM or SNOM)The tip must be ~10 nm from the sample

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r

r

Tip – atom interaction:Van der Waals potential

Potential Energy

Attraction

Repulsion

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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

θ>θc & n1>n2

Calculation of θc for n1=1.5, n2=1.36 → Use Snell’s law:

n1 sinθ1 =n2 sinθ2 ⇒ θ1 =arcsinn2

n1

⎛

⎝⎜⎞

⎠⎟≈650

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Total Internal Reflection Fieldcreates evanescent field

z

I z =I 0e−z d

d=λ

4π n12 sin2 θ1 −n2

2

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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 λ/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, 25:719-723 (1977).

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virometer Brownian diameter

electron microscope diameter

TIRF History II

• What can we measure in this thin excitation field?

• Dynamic movement of labeled biomolecules

Protein dynamics

Vesicle–actin dynamics

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TIRF examples

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Right: Overlay of images. Green: wide field, red: TIRFGregg Gundersen, Columbia University

TIRF examples Cells labeled (tubulin) imaged with wide-field (Center panel) and TIRF illumination.

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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

• 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 273:494-497 (1996).

DAPI

400 450 500 550 600 650 700 750 800

EXCITATION and EMISSION SPECTRA

EXCITATION

EMISSION

Cy2 SpectrumGreen FITC

Cy3 Rhodamine SpectrumOrange

Texas Red Cy3.5

Cy5 Cy5.5

INTENSITY [arb. units]

WAVELENGTH [nm]

5 dyes are sufficient for 24 chromosomes

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Hyper-spectral microscopy II

objective

sample

light source

filter cube

CCD detector

Sagnac interferometer

collimating lens • 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

And the detection of genetic abnormalities…

And recognition…

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FLIM

Arndt-Jovin (1979):

• 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, 27:87-95 (1979).

n = 1

2

3

4

t ≈ 10 ns fluorescence lifetime

• 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 lifetimeBodipy TR =

4.85 nsNile Red = 2.71 ns

0%

20%

40%

60%

80%

100%

0 2 4 6 8 10

time [ns]

excited electrons [%]

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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

Steady-state intensity image

Time-resolved intensity image