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Fluorescence Workshop UMN Physics June 8-10, 2006 Steady-State Fluorometer Joachim Mueller

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Fluorescence Workshop UMN Physics June 8-10, 2006 Steady-State Fluorometer Joachim Mueller Fluorescence Workshop UMN Physics June 8-10, 2006 Steady-state Fluorometer Joachim Mueller Instrumental Setup, light sources, wavelength selection, detectors, polarizers, corrections, inner filter effect ISS PC1 (ISS Inc., Champaign, IL, USA) Fluorolog-3 (Jobin Yvon Inc, Edison, NJ, USA ) QuantaMaster (OBB Sales, London, Ontario N6E 2S8) Fluorometer Components Excitation Polarizer Sample Emission Excitation Wavelength Light Source Polarizer Selection Emission Wavelength Computer Selection Fluorometer: DetectorThe Basics Note: Both polarizers can be removed from the optical beam path OPTICAL LAYOUT: PTI Model QM-4 1 Lamp housing 2 Adjustable slits 3 Excitation Monochromator 4 Sample compartment 5 Baffle 6 Filter holders 7 Excitation/emission optics 8 Cuvette holder 9 Emission port shutter 10 Excitation Correction 11 Emission Monochromator 12 PMT detector Light Sources Lamp Light Sources Xenon Arc Lamp Profiles 1. Xenon Arc Lamp (wide Ozone Free range of wavelengths) Visible UV 2. High Pressure Mercury Lamps (High Intensities but concentrated in specific lines) Mercury-Xenon Arc Lamp Profile 3. Mercury-Xenon Arc Lamp (greater intensities in the UV) 4. Tungsten-Halogen Lamps Detectors Photomultiplier tube (PMT) http://micro.magnet.fsu.edu/primer/flash/photomultiplier Principle of operation: Pictures: Photon Counting (Digital) and Analog Detection time Continuous Current Measurement Signal Photon Counting: Analog: Constant Variable High Voltage Supply Voltage Supply PMT PMT level Anode Current Discriminator = Sets Level TTL Output Pulse averaging (1 photon = 1 pulse) Computer Primary Advantages: Primary Advantage: 1. Sensitivity (high signal/noise) 1. Broad dynamic range 2. Increased measurement stability 2. Adjustable range Wavelength Selection Fixed Optical Filters Tunable Optical Filters Monochromators Long Pass Optical Filters 100 80 Spectral Shape 60 Thickness Size 40 Fluorescence (!?) Transmission (%) 20 0 300 400 500 600 700 800 Wavelength (nm) Hoya O54 More Optical Filter Types… Interference Filters (Chroma Technologies) Broad Bandpass Filter (Hoya U330) 100 80 60 40 20 Transmission (%) 0 300 400 500 600 700 Wavelength (nm) Neutral Density (Coherent Lasers) Monochromator: Principle Diagram of a Czerny-Turner monochromator. Light (A) is focused onto an entrance slit (B) and is collimated by a curved mirror (C). The collimated beam is diffracted from a rotatable grating (D) and the dispersed beam re-focused by a second mirror (E) at the exit slit (F). Each wavelength of light is focused to a different position at the slit, and the wavelength which is transmitted through the slit (G) depends on the rotation angle of the grating. The Inside of a Monochromator Mirrors Grating Nth Order (spectral distribution) Zero Order (acts like a mirror) Order of diffraction 1-st order 0-th order (acts like a mirror) 2-nd order Monochromator Slits The slit size determines the bandpass and the throughput Reducing the slit size leads to … 1. Drop in intensity 2. Narrowing of the spectral selection Changing the Emission Bandpass 1.0 1.0 17 nm 0.8 17 nm 0.8 u) a 0.6 8.5 nm 0.6 6 8.5 nm x10 0.4 4.25 nm 0.4 2.125 nm 4.25 nm 0.2 0.2 Fluorescence ( 2.125 nm 0.0 0.0 520 540 560 580 520 540 560 580 Wavelength (nm) Wavelength (nm) Collected on a SPEX Fluoromax - 2 Typical Background Emission Spectrum Emission Scan: Excitation 300 nm Glycogen in PBS 350 nd Excitation (Rayleigh) Scatter 2 Order Scatter 300 (300 nm) (600 nm) u) 250 a 3 200 Water RAMAN 2nd Order RAMAN x10 150 (334 nm) (668 nm) 100 Fluorescence ( 50 0 200 300 400 500 600 700 Wavelength (nm) Fluorescent Contaminants Raman scatter of water Vibrational modes of water Resonant Stokes Raman scattering Energy for the OH stretch vibrational mode in water (expressed in inverse wavenumbers): 3400 cm-1 Simple formula to calculate the wavelength of the Raman peak: 107 (1) Take the excitation wavelength (say = 587 107 490 nm) and insert in the following − 3400 equation: 490 (2) The result specifies the position of the raman peak in nanometers (i.e. the raman peak is at 587nm for an excitation wavelength of 490nm. Monochromator Polarization Bias Tungsten Lamp Profile Collected on an SLM Fluorometer Wood’s Anomaly Parallel Emission No Polarizer Fluorescence Perpendicular Emission Fluorescence 250 800 250 800 Adapted from Jameson, D.M., Instrumental Refinements in Fluorescence Spectroscopy: Applications to Protein Systems., in Biochemistry, Champaign-Urbana, University of Illinois, 1978. Distortion of Excitation and Emission Spectra Light Source Monochromator Detector Ideal Fluorometer: Efficiency Efficiency Light intensity wavelength wavelength wavelength Emission Correction: Excitation Correction: Correcting the emission spectra can be quite complicated and requires specialized techniques. Luckily, the wavelength and polarization dependent correction factors have been determined and are electronically available (see lab section). A beam splitter diverts a few percent of the excitation light impinging on the sample. This light is measured with a calibrated silicon photodiode (10). Thus the instrument monitors at all times the intensity of the excitation light. Sample Issues Signal Attenuation of the Excitation Light PMT Saturation Excess Emission 30 Fluorescence vs Signal 3 25 x1 6 2 0 6 al 20 LINEAR REGION x10 n 15 1 10 540 560 580 600 620 640 660 680 700 Instrument Sig Wavelength (nm) [Fluorophore] Reduced emission intensity 1. ND Filters 2. Narrow slit widths 3. Move off absorbance peak Polarizers Common Types: The Glan Taylor prism polarizer Glan Taylor (air gap) Two Calcite Prisms Glan Thompson 0 Sheet Polarizers 90 0 Sheet polarizer 90 Two UV selected calcite prisms are assembled with an intervening air space. The calcite prism is birefringent and cut so that only one polarization component continues straight through the prisms. The spectral range of this polarizer is from 250 to 2300 nm. At 250 nm there is approximately 50% transmittance..
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