1.  Cyttron 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.  Cyttron 2 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?

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

4.  Cyttron 4 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? 2a2a z

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

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

7.  Cyttron 7 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 Numerical Aperture & Resolution IV

8.  Cyttron 8 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 Numerical Aperture & Resolution IV

9.  Cyttron 9 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

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

11.  Cyttron 11 ~250 nm ~180 nm ~100 nm ~30 nm Garini et al, Curr Opin Biotech 2005. 16, 3-12 Practice: High-resolution optical methods

12.  Cyttron 12 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

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

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

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

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

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

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

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21.  Cyttron 21

22.  Cyttron 22 NSOM example of a Muscle Tissue Topography Near-field

23.  Cyttron 23 Total Internal Reflection Microscopy Principle: Happens when light hit a surface θ > θ c & n 1 >n 2 Calculation of θ c for n 1 =1.5, n 2 =1.36 → Use Snell’s law:

24.  Cyttron 24 Total Internal Reflection Field creates evanescent field z

25.  Cyttron 25 Why is TIRF interesting? Provides high resolution along z – overcomes wide-field limit Limitation: only measures the surface, Still important for various applications.

26.  Cyttron 26 TIRF microscopy

27.  Cyttron 27 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).

28.  Cyttron 28 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

29.  Cyttron 29 TIRF examples

30.  Cyttron 30 Right: Overlay of images. Green: wide field, red: TIRF Gregg Gundersen, Columbia University TIRF examples Cells labeled (tubulin) imaged with wide-field (Center panel) and TIRF illumination.

31.  Cyttron 31 Advanced TIRF for single molecule detection Setup: Interference Calibration by moving the slide Cappello, G. Physical Review E 68, 2003.

32.  Cyttron 32 Advanced TIRF : Results

33.  Cyttron 33 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 5 dyes are sufficient for 24 chromosomes

34.  Cyttron 34 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

35.  Cyttron 35 Hyper-spectral microscopy III This, in turn, permits spectral karyotyping And the detection of genetic abnormalities… And recognition…

36.  Cyttron 36 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 lifetime Bodipy TR  = 4.85 ns Nile Red  = 2.71 ns

37.  Cyttron 37 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 imageTime-resolved intensity image