Investigation of biomolecular systems Spectroscopies and microscopies

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1 Living cell: ensembles of molecular machines Investigation of biomolecular systems Spectroscopies and microscopies Crawling keratinocyte Microtubule dynamic instability Vesicle transport with kinesin Protein synthesis on the ribosome Methods luminescence Spectroscopy: -interaction between electomagnetic radiation and matter -absorption; fluorescence (FRET); NMR Microscopy: -image formation about objects invisible to the eye -getting around the Abbe criterion Relaxation from excited state followed by light emission Radiation emitted by matter in excess of thermal emission Cold light Processes of fluorescence and phosphorescence

2 Simplified steps of luminescence Fate of absorbed energy Energy absorbance Excitation (stepping to higher energy levels) Internal conversion (heat) k ic k Q Fluorescence quenching Emission of radiation De-excitation (relaxation to ground state) Intersystem crossing S T Fluorescence (ns) Phosphorescence (ms) k isc k f ENERGY k FRET FRET Radiative or non-radiative transitions! Fluorescence Resonance Energy Transfer In general: Occurs by non-radiative dipole-dipole interaction between an excited donor and an proper acceptor molecule under certain conditions (spectral overlap and close distance). Fluorescence Resonance Energy Transfer (FRET): if the participants of the transfer are florophores. - D + FRET Acceptor emission contributes to the relaxation of the excited donor molecule. h h E ~ k FRET ~ 1/R 6 R - A + h

3 Conditions of FRET Distance dependence of FRET Fluorescent donor and acceptor molecules. The distance (R) between donor and acceptor molecules is 2-10 nm! Overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. E = R 0 6 R R 6 E Förster-distance (Distance at which transfer efficiency (E) is 0.5) Fl intenzitás ill. OD Actual distance between fluorophores Hullámhossz (nm) R 0 Applications of FRET Moleculáris ruler: distance measurement on the nm (10-9 m) scale. High sensitivity! Applications: Measurement of interactions between molecules. Measurement of structural changes on molecules. MRI Magnetic Resonance Imaging

4 MRI is a revolutionary device MRI is a non-invasive tomographic method Nuclear Magnetic Resonance Imaging: Basic principle atomic nuclei with nuclear spin: elementary magnets EXCITATION PULSE B 0 B 0 B 0 Atomic nuclei have mass: m proton = 1, g COIL Atomic nuclei carry angular momentum: L = ll+ ( 1) l = spin quantum number B 0 Cryogenic magnet Proton transmits a radiofrequency electromagnetic wave (yellow) after excitation by an RF pulse (red) Otto Stern Atomic nuclei carry charge: q proton = 1, C Radiofrequency coil Gradient coil Water molecule Image Processing (2D-FFT) W. Gerlach Atomic nuclei possess magnetic moment: μ i = L Cross-section of an NMR scanner = gyromagnetic ratio L = angular momentum

5 Nuclear MAgnetic Resonance (NMR) Nuclear Magnetic Resonance: Spin Precession In absence of magnetic field: random orientation of elementary magnets In magnetic field: elementary magnets orient B 0 E parallel energy levels split -1/2 Precession or Larmor frequency: 0 = B 0 f Larmor = 2 B 0 E=hf=μB0 +1/2 Edward Purcell, 1946 Felix Bloch, 1946 antiparallel B 0 B Net magnetization Due to spin access in different energy states Excitation Using radio frequency radiation Low energy state parallel in case of proton Resonance condition: Larmor frequency B 0 M Ratio of magnetic spins in high- (antiparallel) and low-energy (parallel) states: μ i B 0 = magnetic field M = net magnetization N antiparallel N parallel = e E k B T M Boltzmann distribution B 0 = magnetic field M = net magnetization B 1 = irradiated radio frequency wave High energy state antiparallel in case of proton

6 Spin-lattice relaxation T1 or longitudinal relaxation Spin-spin relaxation T2 or transverse relaxation M z M xy free induction decay (FID) T1 relaxation time: depends on interaction between elementary magnet (proton) and its environment t T2 relaxation time: depends on interaction between elementary magnets (protons) t Spin-spin relaxation T2 or transverse relaxation Repetitive pulses of excitation and subsequent relaxation: spin-echo sequence The spin-echo experiment Excitation pulse (90 ) Refocusing pulse (180 ) Excitation pulse (90 ) Refocusing pulse (180 ) TURN BACK!! Erwin Hahn, 1949 Signal T 2* T 2 Time

7 Contrast in MR images is determined by the interaction of spin systems Nuclear Magnetic Resonance Imaging: Two important relaxation mechanisms Hydrogen bridges Spin-lattice relaxation T1 Spin-spin relaxation T2 FREE WATER M Bloembergen Pound Purcell High mobility t B 0 B 0 + INTERMEDIATE LAYER M O - - O C C C N BOUND LAYER Protein, polymer, membrane Low mobility M t Restoration of longitudinal magnetization Energy transfered to lattice (phonons) Entropy increases Repopulation of spins between spin energy levels time Dephasing of transverse magnetization Energy transferred between spins No entropy change of total spin system No repopulation of spins between spin energy levels time t Interactions with magnetic field fluctuations at Larmor frequency Interactions with magnetic field fluctuations at low frequency Nuclear Magnetic Resonance Imaging: Basic principle EXCITATION PULSE From nuclear magnetic resonance signal to Magnetic Resonance Imaging B 0 B 0 B 0 COIL B 0 Cryogenic magnet Proton transmits a radiofrequency electromagnetic wave (yellow) after excitation by an RF pulse (red) Radiofrequency coil Gradient coil Cross-section of an NMR scanner Water molecule Image Processing (2D-FFT)

8 First NMR experiments in vivo MRI: Net magnetization of the human body is generated Downstate Medical Center - Brooklyn, 1972 = B Resonance condition fulfilled Raymond V. Damadian First MRI scan 1970: detection of lengthened relaxation times in cancerous tissues 1972: theoretical development of human in vivo 3D NMR 1977: first human MRI image Inhomogeneous magnetic field Indomitable Spatial encoding of the NMR signal: Imaging gradients X-gradient coil Spatial encoding of the NMR signal is based on frequency changes in the precession y-coil z-coil x-coil Gradient coils Y-gradient coil RF COIL patient Z-gradient coil Fourier transform IMPORTANT NOTE: The magnetic field is always in the Z-direction

9 Spatial encoding: Slice selection NMR scanner with backprojection = B B z Paul Lauterbur, 1973 Illinois Peter Mansfield, 1973 Nottingham 1.52 T GRADIENT COILS f = 64.8 MHz 64.8 MHz Nature 242, (1973), Nobel prize for physiology and medicine (Lauterbur & Mansfield) in 2003 NMR scanner with 2D Fourier transformation The first MRI scanners... Richard Ernst, 1974 Zürich Nobel price for chemistry in 1991 Interventional MRI unit Open MRI unit and recent ones Mobile MRI unit

10 MRI Imaging: Color resolution (contrast) based on spin density and relaxation times MRI: Image manipulation I T1-weighing Proton densityweighing T2-weighing Reslicing in perpendicular plane MRI: Image manipulation II Anatomical imaging: Multiple sclerosis Proton density (sagital) Spatial projection ( volume rendering ) Proton density (transverse) T2 weighted (transverse) T1 weighted With contrast agent

11 Anatomical imaging: Oncology Anatomical imaging Bone and soft tissue T2 weighted (cyst) Protondensity (Brain metastasis) T1 weighted with contrastagent (Breast carcinoma) Rheumatoid arthritis knee Rheumatoid arthritis whrist T2 weighted (chondrosarcoma) T2 weighted (torn ligaments) T2 weighted (hernia) T2 weighted (cervix carcinoma) T2 weighted (prostate tumor) Osteoporosis (femur) There is more to MRI than anatomical imaging... MRI: Non-invasive angiography Image slice 2008 saturated spins blood flow In research phase unsaturated spins 1972 First NMR images State of the art 3D images dynamic images sharp image resolution quantitative imaging cell-specific contrast agents hyperpolarized MRI in vivo spectroscopy functional imaging multimodality imaging

12 MRI: Non-invasive angiography MRI movie Based on high time resolution images arteria carotis Circulus arteriosus Willisii Opening and closing of aorta valve Functional MRI (fmri) High time resolution images recorded synchronously with physiological processes Superposition of MRI on other information (PET) Effect of light pulses on visual cortex

13 Superposed MRI and PET sequence Microscopy PET activity: during eye movement Volume rendering Image formation in the light microscope Possibilities of improving microscope resolution 1. Improving the parameters of the Abbe formula Due to diffraction: image of a point object is an Airy disk Smallest resolved distance (Abbé): d = 0.61 n sin 2. Conversion of the resolution problem into a positioning problem 3. Non-diffraction-limited imaging

14 1. Reduction of wavelength: Electron microscopy 1. Electron as wave, De Broglie (1924): = h mv =wavelength h=planck s constant m=electron mass v=electron travel velocity The electron microscope Source: elektron gun Wehnelt cylinder: focusing electrode 2. Deflecting the electron beam: with magnetic lens F = ebv e sin F=force on electron e=electron charge B=magnetic induction V e =electon velocity =angle between electron path and magnetic field Cathode (tungsten filament) Anode pinhole Upper pole Magnetic lens: electron beam spirals through it. Cavity Electron beam Gap Lower pole Coil Ferromagnetic cover Large vacuum: 10-2 Pa Resolution and contrast in the electron microscope 3. Conversion of resolution problem into positioning problem A. Theoretical resolution: Modified Abbe-formule (for small angles) Based on electon velocity ( km/s), d=0.005 nm B. Real resolution: limited by small NA, ~0.1 nm. Because of small NA, depth of focus is large (several μm). C. Practical resolution in biological samples: 1/10 of section thickness. D. Contrast mechanism: electron diffraction Contrast enhancement: by electron dense dyes d = CD63, lysosome transmembrane protein Cryo-electron microscopy, particle-analysis image reconstruction

15 3. Non-diffraction-limited microscopy Atomic Force Microscopy, AFM Atomic Force Microscope: AFM Oxygen atoms on the surface of rhodium crystal bacteriorhodopsin DNA photodetector cantilever laser Richard P. Feynman: There is plenty of room at the bottom December 29, 1959 Unit of nanoworld: 1 nanometer sample DNA titin AFM Foundations Image formation by scanning Imaging errors photodetector cantilever laser 2 m Oscillating drive signal cantilever Detected signal sample Height contrast Amplitude contrast Phase contrast

16 Beta-amyloid fibrils DNA Red blood cells (smear) Fibrin network ATP-synthase Manipulation of molecules with AFM Single-molecule force spectroscopy Force sawteeth: non-cooperative unfolding of mechanically stable domains 500 Force (pn) Force (pn) Extension ( m) Extension (nm)

17 Nanoindentation, nanolithography Lithos: stone, graphein: to draw photodetector laser Pablo Picasso: "Don Quixote" drawn in polycarbonate surface cantilever surface 1 μm