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Cyttron NSOM Lecture A Surface Imaging Method Prof. Ian T. Young Quantitative Imaging Group Department of Imaging Scienc

Cyttron NSOM Lecture A Surface Imaging Method Prof. Ian T. Young Quantitative Imaging Group Department of Imaging Science & Technology Delft University of Technology. Ernst Abbe, 1873. 500 nm. 200 nm. Theory: Optical resolution is limited.

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Cyttron NSOM Lecture A Surface Imaging Method Prof. Ian T. Young Quantitative Imaging Group Department of Imaging Scienc

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

  2. Ernst Abbe, 1873 500 nm 200 nm Theory: Optical resolution is limited • Methods that are based on lenses have limited spatial resolution • Where does this result originate?

  3. Basic Concepts - Wave Optics Interference Diffraction Christiaan Huygens – Treatise on Light

  4. 2a z 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?

  5. 2a 2b Intensity on screen Numerical Aperture & Resolution II What is the intensity distribution for a 2-D aperture  I(x, y, z)? Sketching the result:

  6. Numerical Aperture & Resolution III Where are the zeroes of the intensity function? Adding one final substitution & approximation: Gives: For air, n = 1:

  7. r [nm] with NA = 0.3,  = 500 nm 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 

  8. r [nm] with NA = 0.3,  = 500 nm 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 

  9. r [nm] with NA = 1,  = 500 nm 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 

  10. r [nm] with NA = 0.3,  = 500 nm 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:

  11. 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

  12. ~50 nm High intensity Low intensity How can we overcome the diffraction limit?? Completely different approach: NEAR FIELD Measure VERY CLOSE to tip ~10 nm

  13. Example of a near-field tip 50 nm hole

  14. 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)

  15. Piezoelectric motors Material (example): Perovskite-type lead zirconate titanate (PZT). Different schemes: single/multi layers high/low voltage

  16. Near-field microscope: feedback mechanism Tuning fork Optical probe & quadrant detector

  17. psd laser laser optical fiber Piezo 3-axis motor tip sample collection optics detector Near-field scanning optical microscope(NSOM or SNOM) The tip must be ~10 nm from the sample

  18. Tip – atom interaction:Van der Waals potential Potential Energy r Repulsion Attraction r

  19. NSOM working modes: Non-contact mode Contact mode Tapping mode

  20. NSOM example of a Muscle Tissue Topography Near-field

  21. 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:

  22. Total Internal Reflection Fieldcreates evanescent field z

  23. Why is TIRF interesting? Provides high resolution along z – overcomes wide-field limit Limitation: only measures the surface, Still important for various applications.

  24. TIRF microscopy

  25. 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, 25:719-723 (1977).

  26. Vesicle–actin dynamics Protein dynamics electron microscope diameter virometer Brownian diameter TIRF History II • What can we measure in this thin excitation field? • Dynamic movement of labeled biomolecules

  27. TIRF examples

  28. 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

  29. Advanced TIRF for single molecule detection Setup: Interference Calibration by moving the slide Cappello, G. Physical Review E 68, 2003.

  30. Advanced TIRF : Results

  31. 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 273:494-497 (1996).

  32. Sagnac interferometer CCD detector collimating lens light source filter cube objective sample 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

  33. This, in turn, permits spectral karyotyping And the detection of genetic abnormalities… And recognition… Hyper-spectral microscopy III

  34. 4 3 2 Bodipy TR = 4.85 ns Nile Red = 2.71 ns n = 1 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, 27:87-95 (1979).

  35. Steady-state intensity image Time-resolved intensity image FLIM • There are several ways to measure this phenomenon: • Sinusoidal light source modulation (now with LEDs!) • Pulse method • Gated method • PRBS light source modulation

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