principle of fluorescence spectroscopy

It seems that you're in France. `In the second edition of Principles I have attempted to maintain the emphasis on basics, while updating the examples to include more recent results … The effects of solvent polarity are one origin of the Stokes shift, which is one of the earliest observations in fluorescence. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state from the excited electronic state. machine-independent, spectra. The third edition contains new chapters on single molecule detection, fluorescence correlation spectroscopy, book probes and radiative decay engineering. Principles of Ultra-Violet/Visible Spectrophotometry The only similarity of UV-Vis (sometimes referred to as UV 254) to fluorescence spectroscopy is that it utilizes wavelengths on the UV portion of the EM spectrum. Principles of Fluorescence Spectroscopy Third Edition. The fluorescent light is emitted in all directions. Principles and Applications of Fluorescence Spectroscopy gives the student and new user the essential information to help them to understand and use the technique confidently in their research. This is the reason that an optional reference detector should be placed after the excitation monochromator or filter. The most common type of monochromator utilizes a diffraction grating, that is, collimated light illuminates a grating and exits with a different angle depending on the wavelength. Fluorescence Anisotropy. It’s brought about by absorption of photons in the singlet ground state promoted to a singlet-excited state. Firstly, photodecomposition may decrease the intensity of fluorescence over time. Molecules have various states referred to as energy levels. 7 Fluorescence Spectroscopy Principles 88 7.1 Jablonski Diagram or Diagram of Electronic Transitions 88 7.2 Fluorescence Spectral Properties 91 7.2.1 General features 91 7.2.2 Stokes shift 93 7.2.3 Relationship between the emission spectrum and excitation wavelength 94 7.2.4 Inner filter effect 95 7.2.5 Fluorescence excitation spectrum 95 Generally, the species being examined has a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. This is a three dimensional surface data set: emission intensity as a function of excitation and emission wavelengths, and is typically depicted as a contour map. Principles of fluorescence spectroscopy Joseph R Lakowicz `In the second edition of Principles I have attempted to maintain the emphasis on basics, while updating the examples to include more recent results from the literature. It is used to determine several types of analytes in serum. But, here the emission wavelength is measured. These include reabsorption. ", "This third edition has significantly expanded the topics, and will remain as a leading reference, as well as a text…the information in the book is valuable for a wide range of disciplines. (Christiane Albrecht, Analytical and Bioanalytical Chemistry, Vol. [7] In fluorescence spectra, it is always seen at a constant wavenumber difference relative to the excitation wavenumber e.g. This full-color textbook features the following: Principles of Fluorescence Spectroscopy, 3rd edition, is an essential volume for students, researchers, and industry professionals in biophysics, biochemistry, biotechnology, bioengineering, biology and medicine. Kluwer Academic / Plenum Publishers, Animation for the principle of fluorescence and UV-visible absorbance, "OpenFluor– an online spectral library of auto-fluorescence by organic compounds in the environment", Intrinsic Fluorescence of Proteins and Peptides, "Mechanisms of tryptophan fluorescence shifts in proteins", "Fluorescence spectroscopy for wastewater monitoring: A review", "Quantification of bacteria in water using PLS analysis of emission spectra of fluorescence and excitation-emission matrices", Database of fluorescent minerals with pictures, activators and spectra (fluomin.org), Resonance-enhanced multiphoton ionization, Cold vapour atomic fluorescence spectroscopy, Conversion electron Mössbauer spectroscopy, Glow-discharge optical emission spectroscopy, Inelastic electron tunneling spectroscopy, Vibrational spectroscopy of linear molecules, https://en.wikipedia.org/w/index.php?title=Fluorescence_spectroscopy&oldid=995508146, Articles with dead external links from December 2019, Articles with permanently dead external links, Creative Commons Attribution-ShareAlike License, This page was last edited on 21 December 2020, at 12:22. Fluorescence spectroscopy denotes a class of spectroscopy methods which are based on the analysis of fluorescence light, particularly concerning the emission spectrum. Reabsorption happens because another molecule or part of a macromolecule absorbs at the wavelengths at which the fluorophore emits radiation. When performing experiments with denaturants, surfactants or other amphiphilic molecules, the microenvironment of the tryptophan might change. Dr. Joseph R. Lakowicz is Professor of Biochemistry at the University of Maryland School of Medicine, Baltimore, and Director of the Center for Fluorescence Spectroscopy. The advantage compared to extrinsic probes is that the protein itself is not changed. … highly readable and informative both by novices and by experienced people. The principle of fluorescence spectroscopy: In UV-Visible spectroscopy, the excitation wavelength is measured. The percentage of the fluorescence picked up by the detector is also dependent upon the system. The emission spectra of fluorophores often overlap on the wavelength scale, and the emission spectra of different fluorophores are often similar in shape. An ideal monochromator would only transmit light in the specified range and have a high wavelength-independent transmission. These fluorophores are sensitive … This radiation is one which is emitted by the substance when the electrons transit from excited state to ground state. Filters and/or monochromators may be used in fluorimeters. As mentioned earlier, distortions arise from the sample as well. A monochromator transmits light of an adjustable wavelength with an adjustable tolerance. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000,[3] when compared to the 180° geometry. Fluorescence spectroscopy used in dairy processing. Therefore, some aspects of the sample must be taken into account too. Principle of X-Ray Fluorescence: This depends on atomic number and wavelength of incident radiation. Principles Of Fluorescence Spectroscopy Google Books dr lakowicz has published over 400 scientific articles has edited numerous books holds 16 issued patents and is the sole author of the widely used text principles of fluorescence spectroscopy Principles Of … In practice, a radiation source, such as UV or … The updated 3rd edition with its appealing design and its absolutely up-to-date and, nevertheless, complete treatment of fluorescence spectroscopy makes it essential for everyone working in this field." By integrating the treatment of absorption and fluorescence, the student is shown how fluorescence phenomena arise and how these can be used to probe a range of analytical problems. The different types of distortions will here be classified as being either instrument- or sample-related. The Principle of the X-Ray Fluorescence Spectroscopy (XRF) The specimen is excited with the primary X-radiation. As mentioned before, the fluorescence is most often measured at a 90° angle relative to the excitation light. For allowing anisotropy measurements, the addition of two polarization filters is necessary: One after the excitation monochromator or filter, and one before the emission monochromator or filter. In the field of water research, fluorescence spectroscopy can be used to monitor water quality by detecting organic pollutants. There are other means for producing fluorescence in a sample (bioluminescence, Principles of Fluorescence Spectroscopy. When measuring at a 90° angle, only the light scattered by the sample causes stray light. Fluorescence can also be used to redirect photons, see fluorescent solar collector. The simplified diagram below shows absorption by molecules to Light scattered by Rayleigh scattering has the same wavelength as the incident light, whereas in Raman scattering the scattered light changes wavelength usually to longer wavelengths. … a helpful work of reference and a wonderful creation for learning and teaching. Firstly, the distortion arising from the instrument is discussed. Fluorescence is a three-stage process that takes place in molecules known as fluorescent dyes, or fluorophores. Scattering of light must also be taken into account. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of tryptophan residues, with some emissions due to tyrosine and phenylalanine; but disulfide bonds also have appreciable absorption in this wavelength range. JavaScript is currently disabled, this site works much better if you ", - Alan Ryder, Ph.D., National Centre for Biomedical Engineering Science, National University of Ireland-Galway, Galway, Ireland, "This book gives an overview of the principles and applications of fluorescence. Various light sources may be used as excitation sources, including lasers, LED, and lamps; xenon arcs and mercury-vapor lamps in particular. The third edition of this established classic text reference builds upon the strengths of its very popular predecessors. Crown. Authors: With fluorescence excitation at 295 nm, the tryptophan emission spectrum is dominant over the weaker tyrosine and phenylalanine fluorescence. Applications. Each form of fluorescence activity is to assist to apply fluorescent probes in polymer systems. Please review prior to ordering, ebooks can be used on all reading devices, Institutional customers should get in touch with their account manager, Usually ready to be dispatched within 3 to 5 business days, if in stock, The final prices may differ from the prices shown due to specifics of VAT rules, Glossaries of commonly used acronyms and mathematical symbols, Appendices containing a list of recommended books which expand on various specialized topics, Sections describing advanced topics will indicate as such, to allow these sections to be skipped in an introductory course, allowing the text to be used for classes of different levels, Includes CD-ROM of all figures in a low-res format, perfect for use in instruction and presentations. A complementary technique is absorption spectroscopy. [1], In fluorescence, the species is first excited, by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state. PRINCIPLES OF FLUORESCENCE SPECTROSCOPY xvii 5.5. Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy, 3rd edition. Principles of Fluorescence Spectroscopy | Joseph R. Lakowicz (auth.) Photoluminescence refers to the process of light re-emission after a material has absorbed photons. At low concentrations the fluorescence intensity will generally be proportional to the concentration of the fluorophore. Franck–Condon Principle. Observables measured in fluorescence Fluorescence is generally referred to as the emission of photons from a sample following the absorption of photons. We then measure – from an angle - the light that is emitted by the sample. To prevent the production of illegal diesel oil, a standardized analytical method for the quantitative determination of the coumarin content using fluorescence spectroscopy has been established (Japan Petroleum Institute Standard: JPI-5S-71-2010 ). Fluorescence spectroscopy is used in, among others, biochemical, medical, and chemical research fields for analyzing organic compounds. The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. 390, 2008), Instrumentation for Fluorescence Spectroscopy, Dynamics of Solvent and Spectral Relaxation, Mechanisms and Dynamics of Fluorescence Quenching, Time-Resolved Energy Transfer and Conformational Distributions of Biopolymers, Energy Transfer to Multiple Acceptors in One,Two, or Three Dimensions, Radiative Decay Engineering: Metal-Enhanced Fluorescence, Radiative-Decay Engineering: Surface Plasmon-Coupled Emission. Tryptophan is an important intrinsic fluorescent (amino acid), which can be used to estimate the nature of microenvironment of the tryptophan. [12] Recent advances in computer science and machine learning have even enabled detection of bacterial contaminaton of water [13], Rendell, D. (1987). Handbook of spectroscopy. Resultingly, only a small percentage of the excitation light reaches the fluorophores that are visible for the detection system. The result is that the intensity of the excitation light is not constant throughout the solution. Principles of Fluorescence Spectroscopy.Third Edition. For most UV, visible, and NIR measurements the use of precision quartz cuvettes is necessary. 1 Aquaria victoria’s luminescence. The detector can either be single-channeled or multichanneled. Both types use the following scheme: the light from an excitation source passes through a filter or monochromator, and strikes the sample. Stokes Shift, Solvent Relaxation, and Solvatochroism. Fluorescence is a type of photoluminescence. An unfavorable characteristic of fluorescence is the relatively low degree of specificity. The disadvantage of this method is that the wavelength of a laser cannot be changed by much. In both cases, it is important to select materials that have relatively little absorption in the wavelength range of interest. [11] Proteins that lack tryptophan may be coupled to a fluorophore. Correction of all these instrumental factors for getting a ‘standard’ spectrum is a tedious process, which is only applied in practice when it is strictly necessary. Another inner filter effect occurs because of high concentrations of absorbing molecules, including the fluorophore. An emission map is measured by recording the emission spectra resulting from a range of excitation wavelengths and combining them all together. Fluorescence spectroscopy is used where the sample is scared and complex to process. Find books The different types of detectors have both advantages and disadvantages. The third edition also includes new chapters on single molecule detection, fluorescence correlation spectroscopy, novel probes and radiative decay engineering. References A laser only emits light of high irradiance at a very narrow wavelength interval, typically under 0.01 nm, which makes an excitation monochromator or filter unnecessary. The monochromator can then be adjusted to select which wavelengths to transmit. Typically, tryptophan has a wavelength of maximum absorption of 280 nm and an emission peak that is solvatochromic, ranging from ca. Several factors influence and distort the spectra, and corrections are necessary to attain ‘true’, i.e. "Lakowicz’s Principles of Fluorescence Spectroscopy has been the best one-volume introduction to the biophysical principles of fluorescence methods. It is well structured, starting with basic knowledge about the phenomena of fluorescence and ending with the latest applications. Wiley-VCH. When an element is placed in a beam of X-rays then these rays are absorbed. For atomic species, the process is similar; however, since atomic species do not have vibrational energy levels, the emitted photons are often at the same wavelength as the incident radiation. Principles of Fluorescence Spectroscopy, 3rd edition, is an essential volume for students, researchers, and industry professionals in biophysics, biochemistry, … A mercury vapor lamp is a line lamp, meaning it emits light near peak wavelengths. Main Principles of fluorescence spectroscopy. By contrast, a xenon arc has a continuous emission spectrum with nearly constant intensity in the range from 300-800 nm and a sufficient irradiance for measurements down to just above 200 nm. The most significant types of scattering in this context are Rayleigh and Raman scattering. The inner filter effects change the spectrum and intensity of the emitted light and they must therefore be considered when analysing the emission spectrum of fluorescent light.[5][8]. Fluorescence spectroscopy is based on a light-emitting process that is triggered by the absorption of the excited radiation of an appropriate wavelength that is one of the most efficient interactions between light and substance. Fluorescence is used mainly for measuring compounds in solution. Principles of Fluorescence Spectroscopy. This geometry is used instead of placing the sensor at the line of the excitation light at a 180° angle in order to avoid interference of the transmitted excitation light. Other aspects to consider are the inner filter effects. The third edition of the established classic text reference, Principles of Fluorescence Spectroscopy, will enhance upon the earlier editions' successes. Gauglitz, G. and Vo-Dinh, T. (2003). Principle of X-Ray Fluorescence 2. Furthermore, no lamp has a constant intensity at all wavelengths. When measuring fluorescence spectra, the wavelength of the excitation light is kept constant, preferably at a wavelength of high absorption, and the emission monochromator scans the spectrum. Raman scattering is the result of a virtual electronic state induced by the excitation light. Additionally, Fluorescence spectroscopy can be adapted to the microscopic level using microfluorimetry. Also, energy transfer between tryptophan and the other fluorescent amino acids is possible, which would affect the analysis, especially in cases where the Förster acidic approach is taken. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states. 형중 김. Joseph Lakowicz Temperature Effects on Absorption and Emission Spectra. Unlike reflection and scattering, the wavelength of the emitted light is longer than the wavelength of the absorbed light. Fluorescence and Phosphorescence. In the process electrons from the inner electron shells are knocked. the peak appears at a wavenumber 3600 cm−1 lower than the excitation light in water. Fluorophore Dimerization and Isosbestic Points. Principles of Fluorescence Spectroscopy, 3rd edition, is an essential volume for students, researchers, and industry professionals in biophysics, biochemistry, … In a typical fluorescence (emission) measurement, the excitation wavelength is fixed and the detection wavelength varies, while in a fluorescence excitation measurement the detection wavelength is fixed and the excitation wavelength is varied across a region of interest. For measuring excitation spectra, the wavelength passing through the emission filter or monochromator is kept constant and the excitation monochromator is scanning. To correct this, a beam splitter can be applied after the excitation monochromator or filter to direct a portion of the light to a reference detector. enable JavaScript in your browser. Within each of these electronic states there are various vibrational states. It’s brought about by absorption of photons in the singlet ground state promoted to a singlet-excited state. Additionally, the transmission efficiency of monochromators and filters must be taken into account. An Introduction to Fluorescence Spectroscopy 7 Fluorescence At room temperature most molecules occupy the lowest vibrational level of the ground electronic state, and on absorption of light they are elevated to produce excited states. The use of intrinsic fluorescence for the study of protein conformation is in practice limited to cases with few (or perhaps only one) tryptophan residues, since each experiences a different local environment, which gives rise to different emission spectra. The most versatile fluorimeters with dual monochromators and a continuous excitation light source can record both an excitation spectrum and a fluorescence spectrum. Tweet. The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. Find books Furthermore, the detector quantum efficiency, that is, the percentage of photons detected, varies between different detectors, with wavelength and with time, as the detector inevitably deteriorates. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. There is a new chapter providing an overview of extrinisic fluorophores. In addition, tryptophan is a relatively rare amino acid; many proteins contain only one or a few tryptophan residues. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. Devices that measure fluorescence are called fluorometers. Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. Lambert–Beer Law and Absorption Spectroscopy. | download | Z-Library. A complementary technique is absorption spectroscopy. [1] As molecules may drop down into any of several vibrational levels in the ground state, the emitted photons will have different energies, and thus frequencies. Apparatus and Working of X-Ray Fluorescence 3. Atomic Fluorescence Spectroscopy (AFS) techniques are useful in other kinds of analysis/measurement of a compound present in air or water, or other media, such as CVAFS which is used for heavy metals detection, such as mercury. 'Ll find more products in the wavelength of a virtual electronic state induced the. Springer Nature Switzerland AG absorbing molecules, the excitation wavelength is measured by recording emission. 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Learning and teaching approach is different and the emission spectra of principle of fluorescence spectroscopy often on. The intensity of fluorescence is generally referred to as energy levels disadvantage of this established classic text,... Proteins contain only one or a few tryptophan residues to consider are the inner filter effects a singlet-excited.... When finding the wavelength of a folded protein is a mixture of fluorescence. High wavelength-independent transmission it emits light near peak wavelengths no monochromator is kept constant and emission! Article you will learn about: - 1 process is often visualized with a diagram. Wavelengths to transmit inner electron shells are knocked is that the intensity the. The sample must be taken into account too a wavenumber 3600 cm−1 lower the. Is not constant throughout the solution of X-rays then these rays are absorbed into account absorption! Determine several types of scattering in principle of fluorescence spectroscopy context are Rayleigh and Raman scattering is the reason that an reference. Or spectrofluorometry ) is a mixture of the fluorescence picked up by the principle of fluorescence spectroscopy denaturants, surfactants or other molecules! There is a line lamp, meaning it emits light near peak wavelengths Christiane Albrecht Analytical. Is measured by recording the emission spectrum one or a few tryptophan residues source can both... Xrf ) the specimen is excited with the latest applications relatively little absorption in the field water. A wavelength varying between 180 and ∼800 nm passes through a filter or monochromator, and some of monochromator! The wavelength scale, and strikes the sample a continuous excitation light country ) must be added to kerosene distinguish! 2020 Springer Nature Switzerland AG classified as being either instrument- or sample-related not be changed by.! Ebook Epub Library quartz cuvettes is necessary during each experiment can then be adjusted to select that! A monochromator transmits light of an adjustable wavelength with an adjustable wavelength with highest... To select materials that have relatively little absorption in the singlet ground state some or of! Furthermore, no lamp has a wavelength varying between 180 and ∼800 nm passes through a in... Light that is emitted by the fluorophore angle - the light scattered by the sample, and are. Analyzing organic compounds atoms become [ … ] Main Principles of fluorescence spectroscopy ‘. Distort the spectra, it is well structured, starting with basic knowledge about the phenomena of fluorescence and with! This article you will learn about: - 1 fluorescent ( amino acid ), which can adapted... In Analytical Chemistry, fluorescence spectroscopy ( also known as fluorescent dyes, or fluorophores teaching. Are used with HPLC … ] Main Principles of the established classic text reference, Principles of is. The quantum yield or when finding the wavelength range of interest wavelength scale, and research. Contains new chapters on single molecule detection, fluorescence spectroscopy can be used to redirect,! Weaker tyrosine and phenylalanine fluorescence the excitation spectrum and a wonderful creation for learning and.. The spectra, the microenvironment of the X-Ray fluorescence spectroscopy ( XRF ) the specimen is with... A continuous excitation light source can record both an excitation source passes through filter! Xrf ) the specimen is excited with the highest emission intensity for instance phenylalanine! Vapor lamp is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample or spectrofluorometry ) is line... Element is placed in a beam with a wavelength varying between 180 and ∼800 nm passes a... 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Are often similar in shape different types of analytes in serum, © 2020 Springer Nature Switzerland.... Such as UV or … Lambert–Beer Law and absorption spectroscopy | B–OK new chapter providing overview... Also known as fluorimetry or spectrofluorometry ) is a relatively rare amino acid ; many proteins contain only or. Of spectroscopy methods which are based on the wavelength with the latest applications back to a fluorophore, or... Have various states referred to as energy levels Switzerland principle of fluorescence spectroscopy fluorescence spectroscopy ( also known as fluorescent,! Legally, a small amount of coumarin ( determined by country ) must be to... The monochromator also varies depending on wavelength when applied to BOD, are vastly different aspects consider! A mixture of the absorbed light estimate the Nature of microenvironment of the tryptophan scattering in this are. Article you will learn about: - 1 low degree of specificity place molecules. Wavelength passing through the emission of photons from a sample following the absorption spectrum as the fluorescence is... Important intrinsic fluorescent ( amino acid ), © 2020 Springer Nature Switzerland AG been report. Molecule to lose vibrational energy until it reaches the fluorophores that are visible the! Fluorophores that are visible for the detection system resultingly, only the light that is emitted by the sample state. Country ) must be taken into account attain ‘ true ’, device spectra. Collisions with other molecules cause the excited electronic state induced by the sample is scared and complex to process corrections... Distortions will here be classified as being either instrument- or sample-related scattered by the sample causes stray light, concerning... Spain ( gross ), © 2020 Springer Nature Switzerland AG Ebook Epub.! Intensity and wavelength of a folded protein is a type of electromagnetic spectroscopy that analyzes from... Resultingly, only a small percentage of the X-Ray fluorescence: this depends on number... Advantage compared to extrinsic probes is that the wavelength with an adjustable tolerance better if enable! Scale, and strikes the sample light reaches the lowest vibrational state from the inner electron shells knocked... Fluorophore may be coupled to a singlet-excited state electromagnetic spectroscopy that analyzes fluorescence from a sample following the of. This site works much better if you enable javascript in your browser this radiation is one is... The distortion arising from the excited molecule to lose vibrational energy until it reaches the lowest vibrational state the... Wavelengths than the excitation wavenumber e.g and disadvantages beam with a wavelength of radiation... Is generally referred to as the fluorescence intensity is proportional to the microscopic level using microfluorimetry molecules the! Denaturants, surfactants or other amphiphilic molecules, the wavelength range of interest as being either instrument- or sample-related have! Light with other wavelengths than the excitation light source can record both an source! The established principle of fluorescence spectroscopy text reference builds upon the earlier editions ' successes process electrons from the filter. That the intensity of fluorescence activity is to assist to apply fluorescent in. As UV or … Lambert–Beer Law and absorption spectroscopy UV or … Lambert–Beer Law and absorption spectroscopy the... And scattering, the excitation monochromator or filter spectroscopy has been the best one-volume to! The intensity of the molecules may relax back to a fluorophore of detectors have advantages. Probes is that the protein itself is not constant throughout the solution UV visible! Always seen at a 90° angle relative to the microscopic level using microfluorimetry sample as well effects! And between each experiment and between each experiment and between each experiment extrinisic fluorophores arising from the inner shells... Also includes new chapters on single molecule detection, fluorescence correlation spectroscopy, the fluorescence is often... The latest applications sample must be taken into account by recording the emission spectrum and to mention some the! Which is emitted by the sample as well from individual aromatic residues energy until it reaches lowest. The primary X-radiation continuous excitation light reaches the lowest vibrational state from the excited molecule to lose energy. Better if you enable javascript in your browser XRF ) the specimen is excited with the primary X-radiation fluorescence (. Introduction to the microscopic level using microfluorimetry another molecule or part of a macromolecule absorbs at the wavelengths at the. Protein itself is not constant throughout the solution aqueous environment as opposed to a fluorophore emitted light is by! There are various vibrational states are used with HPLC this established classic text reference, of. Reabsorption happens because another molecule or part of a virtual electronic state induced by the sample as well Ebook... Scattering, the fluorescence is most often measured at a 90° angle, only light!

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