Principles of. Fluorescence Spectroscopy. Third Edition. Joseph R. Lakowicz. University of Maryland School of Medicine. Baltimore, Maryland, USA. Springer. Third Edition. Joseph R. Lakowicz The first edition of Principles was published in , and tion and fluorescence-correlation spectroscopy are becom-. (c) - page 1 of 8 - Get Instant Access to PDF File: b5b2da Principles Of Fluorescence Spectroscopy By Joseph R. Lakowicz PDF. EBOOK.
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Principles of. Fluorescence Spectroscopy. Third Edition. Joseph R. Lakowicz. University of Maryland School of Medicine. Baltimore, Maryland, USA. Principles of Fluorescence Spectroscopy, 3rd edition, 3rd edition Joseph R. Lakowicz Joseph R. Lakowicz Front Matter. Pages i-xxvi. PDF · Introduction to Fluorescence. Pages PDF · Instrumentation for Fluorescence Spectroscopy. Joseph R. Lakowicz: —Principles of Fluorescence Spectroscopy —. Springer 3rd ed., , XXVI, p., illus., in colour, Hardcover, ISBN.
Principles of Fluorescence Spectroscopy, 3rd edition , 3rd edition Joseph R. Lakowicz The third edition of the established classic text reference, Principles of Fluorescence Spectroscopy, will enhance upon the earlier editions' successes. The third edition also includes new chapters on single molecule detection, fluorescence correlation spectroscopy, novel probes and radiative decay engineering. This full-color textbook features the following: Problem sets following every chapter 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 Principles of Fluorescence Spectroscopy, 3rd edition, is an essential volume for students, researchers, and industry professionals in biophysics, biochemistry, biotechnology, bioengineering, biology and medicine. About the Author: Dr.
A monochromator transmits light of an adjustable wavelength with an adjustable tolerance. 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.
The monochromator can then be adjusted to select which wavelengths to transmit. 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. No monochromator is perfect and it will transmit some stray light , that is, light with other wavelengths than the targeted.
An ideal monochromator would only transmit light in the specified range and have a high wavelength-independent transmission. Furthermore, the fluorescence can also be measured from the front, which is often done for turbid or opaque samples. The single-channeled detector can only detect the intensity of one wavelength at a time, while the multichanneled detects the intensity of all wavelengths simultaneously, making the emission monochromator or filter unnecessary.
The different types of detectors have both advantages and disadvantages. The most versatile fluorimeters with dual monochromators and a continuous excitation light source can record both an excitation spectrum and a fluorescence spectrum.
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. For measuring excitation spectra, the wavelength passing through the emission filter or monochromator is kept constant and the excitation monochromator is scanning.
The excitation spectrum generally is identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption. The different types of distortions will here be classified as being either instrument- or sample-related. Firstly, the distortion arising from the instrument is discussed. As a start, the light source intensity and wavelength characteristics varies over time during each experiment and between each experiment.
Furthermore, no lamp has a constant intensity at all wavelengths. 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. Additionally, the transmission efficiency of monochromators and filters must be taken into account.
These may also change over time. The transmission efficiency of the monochromator also varies depending on wavelength. This is the reason that an optional reference detector should be placed after the excitation monochromator or filter.
The percentage of the fluorescence picked up by the detector is also dependent upon the system. 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. Two other topics that must be considered include the optics used to direct the radiation and the means of holding or containing the sample material called a cuvette or cell.
In both cases, it is important to select materials that have relatively little absorption in the wavelength range of interest. This is the case when measuring the quantum yield or when finding the wavelength with the highest emission intensity for instance. As mentioned earlier, distortions arise from the sample as well.
Therefore, some aspects of the sample must be taken into account too. Firstly, photodecomposition may decrease the intensity of fluorescence over time. Scattering of light must also be taken into account. The most significant types of scattering in this context are Rayleigh and Raman scattering. 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.
Raman scattering is the result of a virtual electronic state induced by the excitation light. From this virtual state , the molecules may relax back to a vibrational level other than the vibrational ground state.
Other aspects to consider are the inner filter effects. These include reabsorption. Reabsorption happens because another molecule or part of a macromolecule absorbs at the wavelengths at which the fluorophore emits radiation. These MLCs display interme the excited state.
In excited singlet states, the electron in the diate lifetimes of hundreds of nanoseconds to several excited orbital is paired by opposite spin to the second microseconds. In this book we will concentrate mainly on electron in the ground-state orbital. Consequently, return to the more rapid phenomenon of fluorescence. The emission rates of fluorescence are cules. Some typical fluorescent substances fluorophores typically s1, so that a typical fluorescence lifetime is are shown in Figure 1.
One widely encountered fluo near 10 ns 10 x s. As will be described in Chapter 4, rophore is quinine, which is present in tonic water. If one the lifetime of a fluorophore is the average time between observes a glass of tonic water that is exposed to sunlight, a its excitation and return to the ground state. It is valuable to faint blue glow is frequently visible at the surface. This consider a 1-ns lifetime within the context of the speed of glow is most apparent when the glass is observed at a right 1 Figure 1.
Figure 1. Structures of typical fluorescent substances. The quinine in tonic water is excited by which, from the circumstances of its occurrence, the ultraviolet light from the sun. Upon return to the ground would seem to originate in those strata which the light state the quinine emits blue light with a wavelength near first penetrates in entering the liquid, and which, if not strictly superficial, at least exert their peculiar power nm.
The first observation of fluorescence from a qui of analysing the incident rays and dispersing those nine solution in sunlight was reported by Sir John Frederick which compose the tint in question, only through a William Herschel Figure 1.
By Sir John hundred times their joint weight of water, and Frederick William Herschel, Philosophical Translation having filtered the solution, pour it into a tall nar of the Royal Society of London It is however strong reflected light from behind. If we look easily and copiously soluble in tartaric acid. Equal down perpendicularly into the vessel so that the weights of the sulphate and of crystallised tartaric visual ray shall graze the internal surface of the acid, rubbed up together with addition of a very little glass through a great part of its depth, the whole water, dissolve entirely and immediately.
It is this of that surface of the liquid on which the light solution, largely diluted, which exhibits the optical first strikes will appear of a lively blue, One notable exception is the group of elements strating that contact with a denser medium has no commonly known as the lanthanides.
These orbitals are shielded effective in producing this superficial colour as a considerable thickness. For instance, if in pour from the solvent by higher filled orbitals. The lanthanides ing it from one glass into another, A fluorescence emission spectrum is a impossible to avoid supposing that we have a plot of the fluorescence intensity versus wavelength highly coloured liquid under our view. Two typical fluores cence emission spectra are shown in Figure 1.
Emission It is evident from this early description that Sir Her spectra vary widely and are dependent upon the chemical schel recognized the presence of an unusual phenomenon structure of the fluorophore and the solvent in which it is that could not be explained by the scientific knowledge of dissolved. The spectra of some compounds, such as pery the time.
To this day the fluorescence of quinine remains lene, show significant structure due to the individual vibra one of the most used and most beautiful examples of fluo tional energy levels of the ground and excited states. Other rescence. Herschel was from a distinguished family of sci compounds, such as quinine, show spectra devoid of vibra entists who lived in England but had their roots in Ger tional structure.
The sensitivity of fluorescence was used in It is interesting to notice that the first known fluo to demonstrate that the rivers Danube and Rhine were rophore, quinine, was responsible for stimulating the devel connected by underground streams.
Some sixty hours later its characteristic green flu interested in monitoring antimalaria drugs, including qui orescence appeared in a small river that led to the Rhine. This early drug assay resulted in a subsequent pro Today fluorescein is still used as an emergency marker for gram at the National Institutes of Health to develop the first locating individuals at sea, as has been seen on the landing practical spectrofluorometer.
Readers interested Many other fluorophores are encountered in daily life. Polynuclear aromatic hydrocar bons, such as anthracene and perylene, are also fluorescent, 1. Some substituted organic The processes that occur between the absorption and emis compounds are also fluorescent.
For example 1,4-bis 5 sion of light are usually illustrated by the Jablonski6 dia phenyloxazolyl benzene POPOP is used in scintilla gram.
Jablonski diagrams are often used as the starting tion counting and acridine orange is often used as a DNA point for discussing light absorption and emission. Jablon stain. Pyridine 1 and rhodamine are frequently used in dye ski diagrams are used in a variety of forms, to illustrate var lasers. Numerous additional examples of probes could be pre These diagrams are named after Professor Alexander sented.
Instead of listing them here, examples will appear Jablonski Figure 1. An overview of ments, including descriptions of concentration depolariza fluorophores used for research and fluorescence sensing is tion and defining the term "anisotropy" to describe the presented in Chapter 3.
In contrast to aromatic organic mol polarized emission from solutions. Professor Alexander Jablonski , circa Courtesy of his daughter, Professor Danuta Frackowiak. Absorption and fluorescence emission spectra of perylene and quinine.
Emission spectra cannot be correctly presented on both Throughout the s and 30s the Depart the wavelength and wavenumber scales. The wavenumber presenta tion is correct in this instance.
Wavelengths are shown for conven ment of Experimental Physics at Warsaw Univer ience. See Chapter 3.
Revised from . During most of this period Jablonski worked both theoretically and Brief History of Alexander Jablonski experimentally on the fundamental problems of Professor Jablonski was born February 26, in photoluminescence of liquid solutions as well as Voskresenovka, Ukraine. In he began his study on the pressure effects on atomic spectral lines in of atomic physics at the University of Kharkov, which gases. A problem that intrigued Jablonski for was interrupted by military service first in the Russian many years was the polarization of photolumi Army and later in the newly organized Polish Army nescence of solutions.
To explain the experimen during World War I.
At the end of , when an inde tal facts he distinguished the transition moments pendent Poland was re-created after more than in absorption and in emission and analyzed vari years of occupation by neighboring powers, Jablonski ous factors responsible for the depolarization of left Kharkov and arrived in Warsaw, where he entered luminescence.
His study in Warsaw was again interrupted in by a world war. From to Jablonski by military service during the Polish-Bolshevik served in the Polish Army, and spent time as a war. In he returned to Poland to first violin at the Warsaw Opera from chair a new Department of Physics in the new to while studying at the university under Nicholas Copernicus University in Torun.
This Stefan Pienkowski. He received his doctorate in beginning occurred in the very difficult postwar for work "On the influence of the change of years in a country totally destroyed by World wavelengths of excitation light on the fluores War II. Despite all these difficulties, Jablonski cence spectra. Professor Jablonski created a spectroscop ic school of thought that persists today through his numerous students, who now occupy posi tions at universities in Poland and elsewhere.
Professor Jablonski died on September 9, More complete accounts of his accomplishments are given in  and . A typical Jablonski diagram is shown in Figure 1. The Figure 1. One form of a Jablonski diagram. At each of these electron trum is typically a mirror image of the absorption spectrum ic energy levels the fluorophores can exist in a number of of the S0 6 S1 transition.
This similarity occurs because vibrational energy levels, depicted by 0, 1, 2, etc. In this electronic excitation does not greatly alter the nuclear Jablonski diagram we excluded a number of interactions that geometry. Hence the spacing of the vibrational energy lev will be discussed in subsequent chapters, such as quenching, els of the excited states is similar to that of the ground state.
The transitions As a result, the vibrational structures seen in the absorption between states are depicted as vertical lines to illustrate the and the emission spectra are similar. Transitions occur in Molecules in the S1 state can also undergo a spin con about s, a time too short for significant displacement of version to the first triplet state T1.
Emission from T1 is nuclei. This is the Franck-Condon principle.
Tran perylene Figure 1. The individual emission maxima sition from T1 to the singlet ground state is forbidden, and and hence vibrational energy levels are about cm1 as a result the rate constants for triplet emission are several apart. At room temperature thermal energy is not adequate orders of magnitude smaller than those for fluorescence.
Molecules containing heavy atoms such as bromine and Absorption and emission occur mostly from molecules with iodine are frequently phosphorescent. The heavy atoms the lowest vibrational energy. The larger energy difference facilitate intersystem crossing and thus enhance phospho between the S0 and S1 excited states is too large for thermal rescence quantum yields. For this reason we use light and not heat to induce fluorescence. Following light absorption, several processes usually 1.
With a few rare excep tions, molecules in condensed phases rapidly relax to the The phenomenon of fluorescence displays a number of gen lowest vibrational level of S1. This process is called internal eral characteristics. Exceptions are known, but these are conversion and generally occurs within s or less. Since infrequent. Generally, if any of the characteristics described fluorescence lifetimes are typically near s, internal in the following sections are not displayed by a given fluo conversion is generally complete prior to emission.
Hence, rophore, one may infer some special behavior for this com fluorescence emission generally results from a thermally pound. The Stokes Shift Return to the ground state typically occurs to a higher excited vibrational ground state level, which then quickly Examination of the Jablonski diagram Figure 1.
Return to that the energy of the emission is typically less than that of an excited vibrational state at the level of the S0 state is the absorption. Fluorescence typically occurs at lower energies reason for the vibrational structure in the emission spectrum or longer wavelengths. This phenomenon was first observed of perylene.
An interesting consequence of emission to by Sir. Stokes in at the University of Cam higher vibrational ground states is that the emission spec- bridge. The source of ultraviolet exci sidered as in some way or other qualitatively different tation was provided by sunlight and a blue glass filter, from the original light.
This filter selec tively transmitted light below nm, which was absorbed Careful reading of this paragraph reveals several by quinine Figure 1.
The incident light was prevented important characteristics of fluorescent solutions. The qui from reaching the detector eye by a yellow glass of wine nine solution is colorless because it absorbs in the ultravio filter. Quinine fluorescence occurs near nm and is let, which we cannot see. The blue color comes only from a therefore easily visible. This is because the quinine solution It is interesting to read Sir George's description of the was relatively concentrated and absorbed all of the UV in observation.
The following is from his report published in the first several millimeters. Hence Stokes observed the inner filter effect.