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Fluorescence spectroscopy and microscopy for biology and medicine Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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Presentation on theme: "Fluorescence spectroscopy and microscopy for biology and medicine Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING."— Presentation transcript:

1 Fluorescence spectroscopy and microscopy for biology and medicine Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING

2 Absorption of light and electronic transitions Basic principles of fluorescence, fluorescence spectra Lifetime of fluorescence and its measurement Quenching of fluorescence and its biological applications Anisotropy of fluorescence and its biological applications Influence of solvent on fluorescence spectra Foerster resonance energy transfer and excimer fluorescence Fluorescent proteins Fluorescence microscopy, confocal and 2-photon microscopy Resolution of fluorescence microscope and its enhancement Fluorescence correlation spectroscopy Photodynamic Therapy Fluorescence spectroscopy and microscopy for biology and medicine CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING Martin Hof, Radek Macháň

3 Basic literature: 1.Lakowicz J.R.: Principles of Fluorescence Spectroscopy, 3rd edn. Springer 2006 2.Hof M., Hutterer R., Fidler V.: Fluorescence Spectroscopy in Biology. Springer Verlag 3.Gauglitz G., Vo-Dinh T.: Handbook of Spectroscopy. Wiley VCH Verlag, Weinheim Prosser V. a kol.: Experimentální metody biofyziky. Academia,Praha Invitrogen Tutorials 6.Becker W.: The bh TCSPC Handbook

4 Why fluorescence? Fluorescent Probe ions electric fields viscosity polarity pH temperature Fluorescence Probes are essentially molecular stopwatches which monitor dynamic events which occur during the excited state lifetime – such as movements of proteins or protein domains it provides information on the molecular environment it provides information on dynamic processes on the nanosecond timescale Also fluorescence is very, very, very sensitive! Work with subnanomolar concentrations is routine while femtomolar and even SINGLE MOLECULE studies are possible with some effort

5 Experimental Systems Actin Mitochondria Nucleus Cell organization and function Multicellular organisms Molecular structure and dynamics GFP in a mouse Biological membrane

6 Instrumentation Microscopes Fluorimeters High throughput platereaders

7 2. Christian Huygens 1692: Developed a wave theory of light A very brief history of the study of light Showed that the component colors of the visible portion of white light can be separated through a prism, which acts to bend the light (refraction) in differing degrees according to the wavelength. Developed a corpuscular theory of light. 1. Sir Isaac Newton 1672: 3. Hans Christian Oersted 1820 Showed that there is a magnetic field associated with the flow of electric current 4. Michael Faraday 1831 Showed the converse i.e. that there is an electric current associated with a change of magnetic field

8 5. James Clark Maxwell: 1865 Published his Dynamical theory of the electromagnetic field which combined the discoveries of Newton, Young, Foucault, Oersted and Faraday into a unified theory of electromagnetic radiation Light consists of electromagnetic transverse waves of frequency and wavelength related by = nc where n is the index of refraction of the medium and c is the speed of the light in vacuum c = 3x10 10 cm/s E B we are interested in interactions of the electric field with the matter

9 6. Max Karl Ernst Ludwig Planck: 1900 Explained the laws of black body radiation by postulating that electromagnetic radiation is emitted at discrete energetic quanta E = h, where Planck constant h = * Js. 7. Albert Einstein: 1905 Explained the explained the photoelectric effect by assuming that light is adsorbed at discrete energetic quanta E = h, photons. 8. Louis de Broglie: 1924 Introduced properties of electromagnetic waves to all particles – the wave-corpuscular dualism of quantum physics. A freely moving particle of momentum p has wavelength =h/p.

10 Wavelength and energy scale, appropriate units X-ray UV Visible light IR Microwave Radio Wavelength nm Frequency Hz Wavenumber cm Energy Kcal Energy eV

11 The optical region of the electromagnetic spectrum UV Visible light IR nm Wavelength nm wavelength << optical elements molecules << wavelength vs. microwave or r.f. techniques the whole molecules sense the same phase of light (vs. X-ray diffraction)

12 Interaction of electromagnetic waves with matter Atoms and molecules described as electric multipoles, first approximation: electric dipole Classical electrodynamics: dipoles oscillate at the frequency of the external electromagnetic field + - Elastic scattering of light

13 Interaction of molecules with photons - quantum description Light exists in form of discrete quanta – photons E = h Atoms and molecules occupy discrete energetic states, which can be found as the solution of Schroedingers equation. electronic states vibrational states rotational states J = 1 microwave region N = 1 IR – VIS region UV – VIS region E Exchange of energy with photons is accompanied by transitions between those states.

14 Interaction of light with matter – overview of processes elastic scattering – no exchange of energy between the molecule and the photon inelastic (Raman) scattering – the photon either gives a part of its energy to the molecule or vice versa absorption or emission of photons by the molecule 1 2 absorption spontaneous emission induced emission induced emission is coherent with incident light spontaneous emission by individual molecules is incoherent scattering is coherent and instantaneous

15 Elastic scattering of light Rayleigh scattering – small molecules (x<0.3) as a point dipole, I sc 4 blue sky, red sunset Larger scatterers – macromolecules, cells, Mie theory for spherically symmetrical scatterers x = 0.07 x = 7

16 Raman scattering C.V. Raman ( ) 1923 theoretically predicted by Adolf Smekal using classical physics 1928 observed by C. V. Raman Stokeselasticanti-Stokes branch of Raman spectrum the photon and the molecule exchange energy the photon is not absorbed: scattering is an instantaneous and coherent v1v1 v2v h +

17 Raman spectrum intensity of Stokes branch is higher by a factor Stokesanti-Stokes

18 Absorption of light I0I0 dx I 1 2 E = hv 0 Nf 1 molecules Nf 2 molecules angle between polarization and D 12, for random orientation of molecules F shape of the spectral line – conservation of energy S small energy approximation – assumes that absorption does not change f 2 /f 1 =exp(E/kT)=(T) electromagnetic energy density M – number of photons N – number of molecules N = c N A S dx

19 Absorption of light I0I0 dx I 1 2 E = hv 0 Nf 1 molecules Nf 2 molecules S the molar extinction coefficient (molar absorptivity) the Lambert Beer law

20 Absorption: measurement The Beer Lambert Law Absorption (Optical Density) = log I o / I = c l l is the path length of the sample (1 cm) Deuterium/ Tungsten Lamp PMT sample PMT reference I IoIo Mono- chromator sample blank Detector a typical sample: a solution in a cuvette the solvent and the reflection from the cuvette walls contribute to the extinction of light relative measurement of absorption

21 Probability HIGH MEDIUM LOW Energy Inter-nuclear distance G S1S1 v 0 v 1 v 2 v 3 v 1 0 v 1 1 v 1 2 v 1 3 Electronic transitions from the ground state to the excited state Probability Wavelength nm

22 Inter-nuclear distance G S1S1 v 0 v 1 v 2 v 3 v 1 0 v 1 1 v 1 2 v 1 3 Electronic transitions from the ground state to the excited state Shaded areas reflects the probability of where the electron would be if it were in that vibrational band Most favored transitions occur From the maximum shaded areas of the ground state To the maximum shaded areas of the excited state

23 Electronic – vibrational spectrum other transitions (other vibrational modes, non- fundamental transitions,…) effect of room temperatureeffect of molecular surroundings

24 The wavelength value of the absorption maximum and the molar absorptivity are determined by the degree of Conjugatation of -bonds Absorption maxima : The importance of conjugation Increasing the number of double bonds shifts the absorption to lower energy molar absorbtivity Wavelength nm N=5 5 pi-bonds, 10 electrons N=4 4 pi-bonds, 8 electrons N=3 3 pi-bonds, 6 electrons

25 As the degree of conjugation increases (i.e the number of electrons involved in the delocalized -orbitals) the absorption energy decreases (>, the energy between the ground and excited state decreases) the absorption becomes more intense (>, increased probability of absorption) Benzene < Naphthalene < Anthracene < naphthacene < pentacene Abs. Max 262nm 275 nm 375 nm 475 nm 580 nm Log (Extinction) Log Extinction Coefficient 275 nm 375 nm 475nm absorption wavelength Increasing the number of aromatic rings increases the absorption maximum

26 Emission of light - Luminescence Luminescence – the excess of light emitted above thermal radiation. The emission follows after the molecule has resided for some time in the excited state. according to excitation mechanism: photoluminescence – absorption of light chemiluminescence – chemical reaction thermoluminescence – heat electroluminescence – electric current … fluorescence phosphorescence photoluminescence – absorption of light

27 Typical sources of luminescence organic molecules (usually with conjugated -bonds) – synthetic fluorophores (fluorescein, rhodamine, …), biological molecules (aromatic amino acids – Trp, Tyr, chlorophyll, …) inorganic crystals (diamond, Si, GaAs, … ) – the spectra depend on the bandgap size, which depends on the size of the crystal (nanocrystals emit in VIS – quantum dots), extreme photostability small inorganic molecules – noble gases (in discharge lamps), N 2 (in lasers, responsible for bluish colour of spark discharges), … quantum dots – same material, different sizes

28 Acknowledgement The course was inspired by courses of: Prof. David M. Jameson, Ph.D. Prof. RNDr. Jaromír Plášek, Csc. Prof. William Reusch Financial support from the grant: FRVŠ 33/119970

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