Fluorescence outline

Information about Fluorescence outline

Published on July 24, 2014

Author: pramodmgu09

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Lecture 15 : Lecture 15 Fluorescence spectroscopy and imaging: Basic principles and sources of contrast Outline for Fluorescence: Outline for Fluorescence Principles of Fluorescence Quantum Yield and Lifetime Fluorescence Spectroscopy Biological Fluorophores Fluorescence Instrumentation Fluorescence Measurements I. Principles of Fluorescence: I. Principles of Fluorescence 1 . Luminescence Emission of photons from electronically excited states Two types of luminescence: Relaxation from singlet excited state Relaxation from triplet excited state I. Principles of Fluorescence: I. Principles of Fluorescence 2 . Singlet and triplet states Ground state – two electrons per orbital; electrons have opposite spin and are paired Singlet excited state Electron in higher energy orbital has the opposite spin orientation relative to electron in the lower orbital Triplet excited state The excited valence electron may spontaneously reverse its spin (spin flip). This process is called intersystem crossing. Electrons in both orbitals now have same spin orientation I. Principles of Fluorescence: I. Principles of Fluorescence 3. Types of emission Fluorescence – return from excited singlet state to ground state; does not require change in spin orientation (more common of relaxation) Phosphoresence – return from a triplet excited state to a ground state; electron requires change in spin orientation Emissive rates of fluorescence are several orders of magnitude faster than that of phosphorescence I. Principles of Fluorescence: I. Principles of Fluorescence 4. Energy level diagram (Jablonski diagram) I. Principles of Fluorescence: I. Principles of Fluorescence 5a. Fluorescence process: Population of energy levels At room temperature (300 K), and for typical electronic and vibration energy levels, can calculate the ratio of molecules in upper and lower states k=1.38*10 -23 JK -1 (Boltzmann’s constant) D E = separation in energy level I. Principles of Fluorescence: I. Principles of Fluorescence 5b. Fluorescence process: Excitation At room temperature, everything starts out at the lowest vibrational energy level of the ground state Suppose a molecule is illuminated with light at a resonance frequency Light is absorbed; for dilute sample, Beer-Lambert law applies where e is molar absorption (extinction) coefficient (M -1 cm -1 ); its magnitude reflects probability of absorption and its wavelength dependence corresponds to absorption spectrum Excitation - following light absorption, a chromophore is excited to some higher vibrational energy level of S 1 or S 2 The absorption process takes place on a time scale (10 -15 s) much faster than that of molecular vibration → “vertical” transition (Franck-Condon principle). So S1 Energy nuclear configuration I. Principles of Fluorescence: I. Principles of Fluorescence 5c. Fluorescence process: Non-radiative relaxation In the excited state, the electron is promoted to an anti-bonding orbital → atoms in the bond are less tightly held → shift to the right for S 1 potential energy curve → electron is promoted to higher vibrational level in S 1 state than the vibrational level it was in at the ground state Vibrational deactivation takes place through intermolecular collisions at a time scale of 10 -12 s (faster than that of fluorescence process) . So S1 I. Principles of Fluorescence: I. Principles of Fluorescence 5d. Fluorescence process: Emission The molecule relaxes from the lowest vibrational energy level of the excited state to a vibrational energy level of the ground state (10 -9 s) Relaxation to ground state occurs faster than time scale of molecular vibration → “vertical” transition The energy of the emitted photon is lower than that of the incident photons So S1 I. Principles of Fluorescence: I. Principles of Fluorescence 6a. Stokes shift The fluorescence light is red-shifted (longer wavelength than the excitation light) relative to the absorbed light ("Stokes shift”). Internal conversion (see slide 13) can affect Stokes shift Solvent effects and excited state reactions can also affect the magnitude of the Stoke’s shift I. Principles of Fluorescence: I. Principles of Fluorescence 6b. Invariance of emission wavelength with excitation wavelength Emission wavelength only depends on relaxation back to lowest vibrational level of S 1 For a molecule, the same fluorescence emission wavelength is observed irrespective of the excitation wavelength So S1 PowerPoint Presentation: I. Principles of Fluorescence 6c. Mirror image rule Vibrational levels in the excited states and ground states are similar An absorption spectrum reflects the vibrational levels of the electronically excited state An emission spectrum reflects the vibrational levels of the electronic ground state Fluorescence emission spectrum is mirror image of absorption spectrum S 0 S 1 v=0 v=1 v=2 v=3 v=4 v=5 v’=0 v’=1 v’=2 v’=3 v’=4 v’=5 I. Principles of Fluorescence: I. Principles of Fluorescence 6d. Internal conversion vs. fluorescence emission As electronic energy increases, the energy levels grow more closely spaced It is more likely that there will be overlap between the high vibrational energy levels of S n-1 and low vibrational energy levels of S n This overlap makes transition between states highly probable Internal conversion is a transition occurring between states of the same multiplicity and it takes place at a time scale of 10 -12 s (faster than that of fluorescence process) The energy gap between S 1 and S 0 is significantly larger than that between other adjacent states → S 1 lifetime is longer → radiative emission can compete effectively with non-radiative emission PowerPoint Presentation: Mirror-image rule typically applies when only S 0 → S 1 excitation takes place Deviations from the mirror-image rule are observed when S 0 → S 2 or transitions to even higher excited states also take place I. Principles of fluorescence: I. Principles of fluorescence 6e. Intersystem crossing Intersystem crossing refers to non-radiative transition between states of different multiplicity It occurs via inversion of the spin of the excited electron resulting in two unpaired electrons with the same spin orientation, resulting in a state with Spin=1 and multiplicity of 3 (triplet state) Transitions between states of different multiplicity are formally forbidden Spin-orbit and vibronic coupling mechanisms decrease the “pure” character of the initial and final states, making intersystem crossing probable T 1 → S 0 transition is also forbidden → T 1 lifetime significantly larger than S 1 lifetime (10 -3 -10 2 s) S 0 S 1 T 1 absorption fluorescence phosphorescence Intersystem crossing I. Principles of fluorescence: I. Principles of fluorescence I. Principles of fluorescence: I. Principles of fluorescence Intensity Wavelength Absorbance DONOR Absorbance Fluorescence Fluorescence ACCEPTOR Molecule 1 Molecule 2 Fluorescence energy transfer (FRET) Intensity Wavelength Absorbance DONOR Absorbance Fluorescence Fluorescence ACCEPTOR Molecule 1 Molecule 2 Non radiative energy transfer – a quantum mechanical process of resonance between transition dipoles Effective between 10-100 Å only Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor II. Quantum yield and lifetime: II. Quantum yield and lifetime Quantum yield of fluorescence, F f , is defined as: In practice, is measured by comparative measurements with reference compound for which has been determined with high degree of accuracy. Ideally, reference compound should have the same absorbance as the compound of interest at given excitation wavelength similar excitation-emission characteristics to compound of interest (otherwise, instrument wavelength response should be taken into account) Same solvent, because intensity of emitted light is dependent on refractive index (otherwise, apply correction Yields similar fluorescence intensity to ensure measurements are taken within the range of linear instrument response 1a. Quantum yield of fluorescence II. Quantum yield and life time: II. Quantum yield and life time Another definition for F f is where k r is the radiative rate constant and S k is the sum of the rate constants for all processes that depopulate the S 1 state. In the absence of competing pathways F f =1 Radiative lifetime, t r , is related to k r The observed fluorescence lifetime, is the average time the molecule spends in the excited state, and it is 1b. Fluorescence lifetime II. Quantum Yield and Lifetime: II. Quantum Yield and Lifetime 2a. Characteristics of quantum yield Quantum yield of fluorescence depends on biological environment Example: Fura 2 excitation spectrum and Indo-1 emission spectrum and quantum yield change when bound to Ca 2+ Fura-2 changes in response to varying [ Ca2+ ] Indo-1 changes in response to varying [ Ca2+ ] II. Quantum yield and lifetime: II. Quantum yield and lifetime 2b. Characteristics of life-time Provide an additional dimension of information missing in time-integrated steady-state spectral measurements Sensitive to biochemical microenvironment, including local pH, oxygenation and binding Lifetimes unaffected by variations in excitation intensity, concentration or sources of optical loss Compatible with clinical measurements in vivo Courtesy of M.-A. Mycek, U Michigan II. Quantum Yield and Lifetime: II. Quantum Yield and Lifetime 3a. Fluorescence emission distribution For a given excitation wavelength, the emission transition is distributed among different vibrational energy levels For a single excitation wavelength, can measure a fluorescence emission spectrum Intensity Emission Wavelength (nm) Exc Emm II. Quantum Yield and Lifetime: II. Quantum Yield and Lifetime 3b. Heisenberg’s uncertainty principle Values of particular pairs of observables cannot be determined simultaneously with high precision in quantum mechanics Example of pairs of observables that are restricted in this way are: Momentum and position Energy and time II. Quantum Yield and Lifetime: II. Quantum Yield and Lifetime 3c. Heisenberg’s uncertainty principle Momentum and position : Energy and time: II. Quantum Yield and Lifetime: II. Quantum Yield and Lifetime 3d. Effect on fluorescence emission Suppose an excited molecule emits fluorescence in relaxing back to the ground state If the excited state lifetime, t is long, then emission will be monochromatic (single line) If the excited state lifetime, t is short, then emission will have a wider range of frequencies (multiple lines) PowerPoint Presentation: Intensity Emission Wavelength (nm) Exc Emm Intensity Emission Wavelength (nm) Exc Emm Large Dt – small DE Small Dt – large DE III. Fluorescence Intensity: III. Fluorescence Intensity 1. Fluorescence intensity expression 2. Fluorescence spectra III. Fluorescence Intensities: III. Fluorescence Intensities 1a. Fluorescence intensity The fluorescence intensity (F) at a particular excitation ( l x ) and emission wavelength ( l m ) will depend on the absorption and the quantum yield: where, I A – light absorbed to promote electronic transition f – quantum yield III. Fluorescence Intensities: III. Fluorescence Intensities 1b. From the Beer-Lambert law, the absorbed intensity for a dilute solution (very small absorbance) where, I o – Initial intensity e – molar extinction coefficient C – concentration L – path length III. Fluorescence Intensities: III. Fluorescence Intensities 1c. Fluorescence intensity expression The fluorescence intensity (F) at a particular excitation ( l x ) and emission wavelength ( l m ) for a dilute solution containing a fluorophore is: where, I o – incident light intensity f – quantum yield C – concentration e – molar extinction L – path length coefficient III. Fluorescence Intensities: III. Fluorescence Intensities 1d. Measured fluorescence intensity If we include instrument collection angle: where, Z – instrumental factor I o – incident light intensity e – molar extinction coefficient C = concentration L – path length III. Fluorescence Intensities: III. Fluorescence Intensities 2a. Fluorescence spectra Emission spectrum Hold excitation wavelength fixed, scan emission Reports on the fluorescence spectral profile reflects fluorescence quantum yield , f k ( l m ) III. Fluorescence Intensities: III. Fluorescence Intensities 2b. Fluorescence spectra Excitation spectrum Hold emission wavelength fixed, scan excitation Reports on absorption structure reflects molar extinction coefficient, e ( l x ) III. Fluorescence Intensities: Fluorescence Intensity Emission Wavelength (nm) Fixed Excitation Wavelength (b) Fluorescence Intensity Excitation Wavelength (nm) Fixed Emission Wavelength (a) III. Fluorescence Intensities III. Fluorescence Intensities: III. Fluorescence Intensities 2c. Fluorescence spectra Composite: Excitation-Emission Matrix Good representation of multi-fluorophore solution III. Fluorescence Intensities: Fluorescence Intensity Emission Wavelength (nm) Fixed Excitation Wavelength Excitation Wavelength (nm) Emission Wavelength (nm) Emission spectrum Excitation-emission matrix III. Fluorescence Intensities IV. Biological Fluorophores: IV. Biological Fluorophores 1. Table 2. EEMs of Epithelial cell suspension 3. EEMs of Collagen IV. Biological Fluorophores: IV. Biological Fluorophores Endogenous Fluorophores amino acids structural proteins enzymes and co-enzymes vitamins lipids porphyrins Exogenous Fluorophores Cyanine dyes Photosensitizers Molecular markers – GFP, etc. IV. Biological Fluorophores: IV. Biological Fluorophores Epithelial Cell Suspension Fluorescence intensity excitation-emission matrix: TCL-1 Emission(nm) Excitation(nm) 300 350 400 450 500 550 600 650 700 250 270 290 310 330 350 370 390 410 430 450 470 490 510 530 550 9.767e-001 6.646e+004 1.306e+005 1.947e+005 4.785e+005 1.120e+006 1.761e+006 2.923e+006 9.335e+006 1.575e+007 2.216e+007 Epithelial Cell Suspension Fluorescence intensity excitation-emission matrix FAD NADH Tryp. Courtesy of N. Ramanujam PowerPoint Presentation: Carbohydrates Fatty Acids and Glycerol Amino Acids Acetyl CoA CITRIC ACID CYCLE CoA CO 2 FADH 2 NADH NADH-Q Reductase Cytochrome Reductase Cytochrome Oxidase Q Cytochrome C O 2 FAD NAD + Oxidation of NADH and FADH 2 by O 2 drives synthesis of ATP ELECTRON TRANSPORT Metabolic Indicators: Metabolic Indicators Metabolism Redox Ratio: FAD / (FAD+NADH) Redox ratio ~ Metabolic Rate Collagen I (gel): 0 days Emission [nm] Excitation [nm] 7 days Collagen I (gel) K. Sokolov 39 days IV. Biological Fluorophores: IV. Biological Fluorophores Collagen It is the major extracellular matrix component, which is present to some extent in nearly all organs and serves to hold cells together in discrete units Collagen fluorescence in load-bearing tissues is associated with cross-links, hydroxylysyl pyridoline (HP) and lysyl pyridinoline (LP). Collagen crosslinks are altered with age and with invasion of cancer into the extracellular matrix V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 1. Introduction 2. Components of a spectrofluorometer 3. Description of key components V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 1. Introduction Fluorescence is a highly sensitive method (can measure analyte concentration of 10 -8 M) Important to minimize interference from: Background fluorescence from solvents Light leaks in the instrument Stray light scattered by turbid solutions Instruments do not yield ideal spectra: Non-uniform spectral output of light source Wavelength dependent efficiency of detector and optical elemens V. Fluorescence Instrumentation: V. Fluorescence Instrumentation Illumination source Broadband (Xe lamp) Monochromatic (LED, laser) Light delivery to sample Lenses/mirrors Optical fibers Wavelength separation (potentially for both excitation and emission) Monochromator Spectrograph Detector PMT CCD camera 2. Major components for fluorescence instrument V. Fluorescence Instrumentation: V. Fluorescence Instrumentation Components of the spectrofluorometer (standard fluorescence lab instrument for in vitro samples) Xenon lamp ( > 250 nm ) Excitation and emission monochromator Each contains two gratings to increase purity of the light Automatic scanning of wavelength through motorized gratings Sample compartment Photo multiplier tube PowerPoint Presentation: PMT Xenon Source Excitation Monochromator Emission Monochromator Sample compartment V. Fluorescence Instrumentation 2. Spectrofluorometer schematic V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3a. Xenon light source Continuous output from Xenon: 270-1100 nm Power – typically 200-450 W Lifetime of ~2000 hours Strong dependence on wavelength V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3a. Xenon light source: broad illumination in the near UV-visible range PowerPoint Presentation: PMT Xenon Source Excitation Monochromator Emission Monochromator Sample compartment V. Fluorescence Instrumentation V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3b. Monochromator: only a small range of wavelengths are focused at the exit slit determined by angle of light incident on the diffraction grating Principle of diffraction grating operation V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3b. Monochromator – Spectral Resolution Inversely proportional to product of dispersion (nm/mm) of grating and the slit width (mm) ~ 5 nm sufficient for fluorescence measurements of biological media Signal increases with the slit width V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3b. Monochromator – Stray light Light which passes through monochromator besides that of desired wavelength Double grating monochromator (stray light rejection is 10 -8 – 10 -12 ) but signal is decreased V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3b. Monochromator – Signal efficiency Grating has a wavelength dependent efficiency Can choose the wavelength at which grating is blazed (maximal efficiency) Excitation monochromator should have high efficiency in the UV; emission monochromator should have high efficiency in the visible PowerPoint Presentation: PMT Xenon Source Excitation Monochromator Emission Monochromator Sample compartment V. Fluorescence Instrumentation V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3c. Photomultiplier tube Contains a photocathode: light sensitive material, which yields electrons upon interaction with photons based on photoelectric effect. Electrons are multiplied by a series of dynodes Provides current output proportional to light intensity V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3c. PMT – Linearity response Current from PMT is proportional to light intensity Under high intensity illumination, PMT will saturate (dynamic range); at low intensity, limited by dark noise Excessive light can damage photocathode, resulting in loss of gain and increased dark noise (thermal noise) V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3c. PMT – Quantum efficiency Quantum efficiency gives the photon to electron conversion efficiency Highly dependent on wavelength V. Fluorescence Instrumentation: V. Fluorescence Instrumentation 3c. Key components – Noise Dark current – Noise due to thermal generation; increases with temperature and high voltage Shot noise – proportional to the square root of the signal VI. Fluorescence Measurements: VI. Fluorescence Measurements VI. Fluorescence measurements 1. Instrument non-uniformities 2. Excitation wavelength calibration 3. Emission wavelength calibration 4. Setup parameters for emission spectrum 5. Routine experimental procedure 6. Collection geometry 7. Blank scans 8. Typical fluorescence spectrum VI. Fluorescence Measurements: VI. Fluorescence Measurements 1a. Ideal spectrofluorometer Light source must yield constant photon output at all wavelengths Monochromator must pass photons of all wavelengths with equal efficiency The PMT must detect photons of all wavelengths with equal efficiency VI. Fluorescence Measurements: VI. Fluorescence Measurements Light Intensity Wavelength LIGHT SOURCE Efficiency Wavelength MONOCHROMATOR Efficiency Wavelength PMT VI. Fluorescence Measurements: VI. Fluorescence Measurements 1b. Distortions in excitation and emission spectra Light intensity from light source is a function of wavelength Monochromator efficiency is a function of wavelength The PMT does not have equal efficiency at all wavelengths VI. Fluorescence Measurements: VI. Fluorescence Measurements 1c. Calibration Correction of variations in wavelength of Xenon lamp and excitation monochromator Need to do when measuring excitation spectra or emission spectra at multiple excitation wavelengths Correction of emission monochromator and PMT Need to do when measuring emission spectra VI. Fluorescence Measurements: VI. Fluorescence Measurements 2a. Excitation wavelength calibration Excitation spectra are distorted primarily by the wavelength dependent intensity of the light source Can use reference photodetector (calibrated) next to sample compartment to measure fraction of excitation light The measured intensity of the reference channel is proportional to the intensity of the exciting light I o F Sample Compartment To PMT To Reference Photodiode VI. Fluorescence Measurements: VI. Fluorescence Measurements 2b. Effect of excitation wavelength calibration VI. Fluorescence Measurements: VI. Fluorescence Measurements 3a. Emission wavelength calibration Need correction factors Measure wavelength dependent output from a calibrated light source Standard lamps of known and calibrated spectral outputs are available from the National Institute of Standards and Testing (NIST) This measurement is typically done by factory; it is difficult to perform properly with commercial fluorimeter To PMT Sample Compartment Calibrated Lamp VI. Fluorescence Measurements: VI. Fluorescence Measurements 3b. Emission wavelength calibration procedure Measure intensity versus wavelength (I( l )) of standard lamp with spectrofluorometer Obtain the spectral output data (L( l )) provided for the lamp Correction factor: S( l ) = L( l )/ I( l ) Multiply emission spectrum with correction factor VI. Fluorescence Measurements: VI. Fluorescence Measurements 3c. Emission wavelength calibration curve VI. Fluorescence Measurements: VI. Fluorescence Measurements 5. Routine experimental procedures Check wavelength calibration of excitation monochromator Check wavelength calibration of emission monochromator Check throughput of spectrofluorometer Xe lamp scan Hg lamp spectrum scan Rhodamine standard scan VI. Fluorescence Measurements: VI. Fluorescence Measurements 6a. Collection geometry in sample compartment Front face – collection is at a 22 degree angle relative to the incident beam; appropriate for an optically absorbing / scattering sample; more stray light Right angle – collection is at a right angle to the incident light; appropriate for optically transparent sample; less stray light I o F I o F Front Face Right Angle VI. Fluorescence Measurements: VI. Fluorescence Measurements 6c. Features of right angle illumination Appropriate for optically transparent sample At high optical densities, signal reaching detector will be significantly diminished VI. Fluorescence Measurements: VI. Fluorescence Measurements 6d. Features of front face illumination Appropriate for an optically absorbing / scattering sample At high optical densities, light is absorbed near the surface of the cuvette containing the absorber; therefore fluorescence is detectable Fluorescence independent of concentration at high optical densities VI. Fluorescence Measurements: VI. Fluorescence Measurements 7. Blank scan Blank is identical to sample except it does not contain fluorophore Measuring the fluorescence of these samples allows the scattering (Rayleigh and Raman) to be assessed In addition, such samples can reveal the presence of fluorescence impurities, which can be subtracted VI. Fluorescence Measurements: VI. Fluorescence Measurements 8. Typical fluorescence emission spectrum at 340 nm excitation (the different components) 0 500000 1000000 1500000 2000000 2500000 3000000 300 350 400 450 500 550 600 Wavelength (nm) Fluorescence Intensity (a.u.) Raman Rayleigh ( l exc = l emm ) Fluorescence

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