3 1 Ctirad Hofr Instrumentation for the detection of absorption and fluorescence Fluorescence methods in life sciences: a journey from a molecule to cell Overview • What light source and when should we use? • How to select light of a given wavelength? • How can we detect light? • What is basic setup of all instruments that measure fluorescence? 3 2 3 3 Absorption • Light is absorbed in material • For the absorption of monochromatic light • Lambert-Beer law: The absorbance is directly proportional to the concentration and thickness of the solution layer lc II ⋅⋅− ⋅= ε 100 I I lcA 0 10log=⋅⋅= ε ε = molar extinction coefficient, c - concentration, l - optical path-length 3 4 SpectroPHOTOmeter • Instrument for measuring the amount of light absorbed by the sample Absorbance is measured at different wavelengths The result is an absorption spectrum of the sample Light source Slit Selection of wavelength Sample Detector 3 5 3 6 The light sources and their use • Tungsten lamp (measuring absorption in the visible spectrum) • Deuterium lamp (measuring absorption in the UV spectrum) • Xenon arc lamp (source for time-steady fluorescence) Pulse sources for time-resolved fluorescence • Laser • LD a LED diodes 3 7 Tungsten lamp – a visible spectrum The glass bulb filled with an inert gas. The tungsten filament is inside, which is heated by direct current. It produces a large amount of heat. Only 5-10% of the energy is released as light. 3 8 Deuterium lamp • Low pressure source particularly suitable for UV spectrum(160-400 nm) • Filled with deuterium in gaseous state 3 9 Xenonon lamp 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 250 300 350 400 450 500 550 600 wavelength, nm current,µA Xenon Arc Lamp (XB0), relatively smooth spectrum 220nm – 1000nm - + Provided by HORIBA Jobin Yvon 3 10 LASER • Most of the molecules of the substance must be in the excited state • After absorption of light, the molecule will be stimulated and it emits a photon • Photons subsequently causes the emission of doubling the number of photons • All photons have the same energy and wavelength and the emitted light has the same color – is monochromatic and is also coherent • The substance is continuously supplied with energy, that the molecules are still in the excited state • Photons are reflected inside the space between two mirrors • Photons in pulses passing through partially permeable mirror • The distance between the pulses is given by the size of the space between the mirrors and the speed of the cycle • Spectrum is a line - only one wavelength mirror mirror (partially permeable) pulses Light Amplification by Stimulated Emission of Radiation http://www.edumedia-sciences.com/a392_l2-laser.html 3 11 LD - Laser Diode Thermoelectric cooler Laser diode housing Collimating lens assembly Anamorphic Prism pair Beam Collimation & Temperature Stabilization Provided by HORIBA Jobin Yvon 3 12 LED Light Emiting Diode • low power consumption • high efficiency • sufficiently narrow spectral range 3 13 The light sources and their use • Tungsten lamp (measuring absorption in the visible spectrum) • Deuterium lamp (measuring absorption in the UV spectrum) • Xenon arc lamp (source for time-steady fluorescence) Pulse sources for time-variable fluorescence: • Laser • LD a LED diodes 3 14 Monochromator It uses light dispersion on a grid the wavelength range is selected by the slit Normal to grating Normal to groove face Incident light Beam of dispersed light Width of the groove Blaze angle Reflection angle Dispersed light Slit Provided by HORIBA Jobin Yvonhttp://www.shsu.edu/~chm_tgc/sounds/Czechdir/Grating%20Czech.swf 3 15 Photoelectric effect - - - - - - - - hf = W+ Ek W – the energy required for ejection of the electron Ek - The kinetic energy of a free electron after the ejection Nobel prize A. Einstein, 1921 http://www.youtube.com/watch?v=v5h3h2E4z2Q 3 16 Detector – photomultiplier tube (PMT) Light Detectors: PhotoMultiplier Tube (R928 Side-on) Side window 24 mm2 Photocathode Focusing electrode Electron multipliers (dynodes) Photon Photoelectron Anode Vacuum 10-4 Pa Provided by HORIBA Jobin Yvon Diode array detector • Array of photosensitive diodes • Each diode absorbs light of a specific wavelength range • Advantage: the whole spectrum is scanned simultaneously 3 17 http://www.youtube.com/watch?v=zbTM36_7jIg&feature=related CCD detektor Charge Coupled Device 3 18 a) light = photons (raindrops) hit electrodes and release electrons that are accumulated as charge b) after a certain exposition time (integration time) a partial charge from each pixel is accumulated from each pixel (point at the detector). The charge is transferred gradually in rows to the outer row (register) where the charge is measured c) The charge collected for each pixel is then determined and converted to voltage. Resulting image is created from bright and dark pixels according to voltage values i.e. number of photons that hit the detector in a given point. How CCD works 3 19 3 20 SpektroPHOTOmeter http://www.youtube.com/watch?v=pxC6F7bK8CU Tungsten lamp (VIS) /deuteruim lamp (UV) Slit Grating monochromator Cuvette made of UV/Vis transparent glass Photomultiplier 3 21 Nanodrop http://www.nanodrop.com/LikeItsHotVideo.aspx 3 22 3 23 Nanophotometer 3 24 Geometry in absorbance measurement cuvette from above Incident light Transmitted light 3 25 The range for measuring the absorbance The most accurate area: i.e. the values are the most reliable in the interval ABS = 0.3 - 0.7 Range with linear response: i.e. that ABS = cε l ABS = 0.1 – 1.0 What to watch when measuring? 3 26 3 27 Instruments for measuring the fluorescence 1. Spectrofluorometers – measure the middle signal of the whole sample usually placed in the cuvette or in the microplates well 2. Fluorescence scanners (including microplate readers) – measure the fluorescence of two-dimensional macroscopic objects (electrophoretic gels, blots, chromatograms) 3. Fluorescence microscopes – to observe the fluorescence of two- or three-dimensional microscopic objects 4. Flow cytometers – measure the fluorescence of a large number of individual cells and allow the identification and separation of their subpopulations 3 28 How a mosquito contributed to the development of instruments? 3 29 DetectorXenon lamp Emission monochromator SpectroFLUOROmeter Provided by HORIBA Jobin Yvon 3 30 Filters Advantages in comparison with the monochromator : Cheap, compact, high-efficiency, high-throughput Disadvantages: not adjustable, often with the specific requirements, an instrument can hold a limited umber of fliters (carousel), have internal fluorescence Cut-off or long pass Band-pass Short-pass Clean-up Notch N.D neutral density 3 31 Sources and filters https://www.youtube.com/watch?v=xJGmARfBasU&t=208s 3 32 L-geometry of fluorescence measurements Taken from J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition,Springer, 2006 Cuvette from above Incident excitation light emitted light 3 33 Effect of inner filter The fluorescence intensity at a certain value of the absorbance (usually from about 0.1 Abs) becomes nonlinear. This is caused by the fact that the layers of the sample distant from plane of incidence of the excitation light on the sample are excited by lower light intensity, because part of the excitation light is absorbed by the surface layers. This error appears only in strongly absorbing solutions, but it has to be always considered and corrected for the precision measurements of quantum yields. Taken from J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition,Springer, 2006 3 34 Effect of concentration of the fluorophore to the shape and position of its emission spectrum Taken from J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition,Springer, 2006 High concentration of anthracene causes a reduction of emission signal at lower wavelengths. It is caused by reabsorption of fluorescence by anthracene in the area where absorption and emission spectrum is most overlapped, i.e. in 370 - 400 nm. The largest effect of he concentration on the emission spectrum is observed in fluorophore with a small Stokes shift – a large overlap of absorption and emission spectrum. The figure shows three concentrations of Rhodamine 6G increasing from left to right and the corresponding change in the color of emitted light. This effect is due to reabsorption of shorter wavelengths of emission spectrum. This shifts the whole spectrum to longer wavelengths at higher concentrations of fluorophore. 3 35 Effect of setting the width of slits on the shape of the spectrum Taken from J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition,Springer, 2006 3 36 Common mistakes when preparing fluorescent samples • Fluorophore concentration is too high - Use a dilute solution of Abs under 0.05 • Contamination of solvents or cuvette - For the unknown sample always validate background, ie. measure the fluorescence spectrum of the cuvette itself only with the solvent • Solid particles in solution - Filter the solution 3 37 Documentation system „ Intelligent darkroom“ It allows to determine the local optical absorption and fluorescence. The source of excitation light is usually transilluminator which emits in the UV region. The source of selective excitation light are LED diodes that are on the sides. Emission is monitored by (digital) camera or cooled CCD detector 3 38 Fluorescence scanner • Scans area and observes the dependence of fluorescence emission intensity at a given excitation light • Used for detection of fluorescently labeled and stained molecules after electrophoretic separation 3 39 Fluorescence scanner - scheme Taken from the product documentation of FUJIFILM 3 40 Fluorescence microscope Main difference compared to spectrometers: does not use monochromators, but excitation and emission filter Taken from J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition,Springer, 2006 http://micro.magnet.fsu.edu/primer/java/ lightpaths/fluorescence/index.html 3 41 Microplate reader Taken from the instrument documentation of Biotek Next How to measure steady-state fluorescence? 3 42