* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Document
Astronomical spectroscopy wikipedia , lookup
Preclinical imaging wikipedia , lookup
Fluorescence correlation spectroscopy wikipedia , lookup
Image intensifier wikipedia , lookup
Ellipsometry wikipedia , lookup
Gaseous detection device wikipedia , lookup
Atmospheric optics wikipedia , lookup
Scanning electrochemical microscopy wikipedia , lookup
Thomas Young (scientist) wikipedia , lookup
Nonimaging optics wikipedia , lookup
3D optical data storage wikipedia , lookup
Anti-reflective coating wikipedia , lookup
Nonlinear optics wikipedia , lookup
Night vision device wikipedia , lookup
Scanning joule expansion microscopy wikipedia , lookup
Chemical imaging wikipedia , lookup
Optical tweezers wikipedia , lookup
X-ray fluorescence wikipedia , lookup
Ultrafast laser spectroscopy wikipedia , lookup
Retroreflector wikipedia , lookup
Surface plasmon resonance microscopy wikipedia , lookup
Interferometry wikipedia , lookup
Optical aberration wikipedia , lookup
Magnetic circular dichroism wikipedia , lookup
Ultraviolet–visible spectroscopy wikipedia , lookup
Johan Sebastiaan Ploem wikipedia , lookup
Optical coherence tomography wikipedia , lookup
Harold Hopkins (physicist) wikipedia , lookup
Photon scanning microscopy wikipedia , lookup
Confocal microscopy wikipedia , lookup
Vibrational analysis with scanning probe microscopy wikipedia , lookup
Cyttron NSOM Lecture A Surface Imaging Method Prof. Ian T. Young Quantitative Imaging Group Department of Imaging Science & Technology Delft University of Technology Cyttron 1 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 Cyttron 2 Basic Concepts - Wave Optics • Interference • Diffraction QuickTime™ and a Animation decompressor are needed to see this picture. Christiaan Huygens – Treatise on Light Cyttron 3 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 Cyttron 4 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: sincq sinq q Intensity on screen • Sketching the result: : 1b Cyttron 5 : 1a Numerical Aperture & Resolution III • Where are the zeroes of the intensity function? kxa sinc z x̂ z ka z 2a • Adding one final substitution & approximation: tan a z for small sin tan • Gives: x̂ z 2a 2 sin n• 2NA • For air, n = 1: square aperture round aperture 0.5 0.61 x̂ x̂ NA NA Cyttron 6 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 Cyttron 7 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 Cyttron 8 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 Cyttron 9 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 R .61 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 Cyttron 10 Practice: High-resolution optical methods ~250 nm ~180 nm ~30 nm ~100 nm ~30 nm Cyttron 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 Cyttron 12 Example of a near-field tip 50 nm hole Cyttron 13 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) Cyttron 14 Piezoelectric motors Material (example): Perovskite-type lead zirconate titanate (PZT). Different schemes: single/multi layers high/low voltage Cyttron 15 Near-field microscope: feedback mechanism Tuning fork Cyttron Optical probe & quadrant detector 16 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 Cyttron 17 Tip – atom interaction: Van der Waals potential Potential Energy r Repulsion Attraction Cyttron r 18 NSOM working modes: Non-contact mode Contact mode Tapping mode Cyttron 19 Cyttron 20 Cyttron 21 NSOM example of a Muscle Tissue Topography Cyttron Near-field 22 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 Cyttron n 1 arcsin 2 65 0 n1 23 Total Internal Reflection Field creates evanescent field z I z I 0 ez d d Cyttron 4 n12 sin 2 1 n22 24 Why is TIRF interesting? Provides high resolution along z – overcomes wide-field limit Limitation: only measures the surface, Still important for various applications. Cyttron 25 TIRF microscopy Cyttron 26 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. Cyttron 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). 27 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 Cyttron 28 TIRF examples Cyttron 29 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 Cyttron 30 Advanced TIRF for single molecule detection Setup: Interference Calibration by moving the slide Cyttron Cappello, G. Physical Review E 68, 2003. 31 Advanced TIRF : Results Cyttron 32 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, 33 Cyttron 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 Cyttron 34 • 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… Cyttron 35 FLIM Arndt-Jovin (1979): 4 3 2 n=1 Dt ≈ 10 ns fluorescence lifetime 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 this fluorescence lifetime 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 36 Cyttron 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 Cyttron 37