Phenomenon of Fluorescence

While there is continued growth in the applications of fluorescence, and continued development of instrumentation and fluMescent probe tM hnology, the principles remain the same and need to be understood by the practitioners. H ice, this volume describes the basic phenomenon of flutM escence, so that these principles can be used successfully in basic and applied research. Throughout the book, we have included examples and applicatitms that illustrate the vinci(rtes offluMescence. 

Luminescence is the emission of light from any substance and occurs from electronically excited states. Luminescence is formally divided into two categories, fluorescence and phosphorescence, depending on the nature of the excited state. In excited singlet states, the electron in the 

If the liquid be poured out iitto anodic vessel, die descending stream gleams internally from all iis undulating inequalities, widi die same lively yet deficate Uue colour. dnis dearly demonstrating that i ct with a denser medium has no share in producing diis singular phenomenon. 

The thinnest film of the liquid seems quite as effective in producing this superficial colour as a considerable thickness. For instance, jf in pouring it from one glass into another. the end of the funnd be made to touch the internal surface of the vessd well moistened, so as to spread the descendingsfreamover an extensive surface, the intensity of the colour is such di it is almost impossible to avoid supposing that we have a highly colored liquid under our view. 

On a Case of Superficial Colour presented by a homogeneous liquid internally colourless. By Sir Jedm Frederick William Herschei Philosophical Transactions of the Royal Society of London (1845) 135 143-145. 

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The discovery and detailed investigations of the phenomenon of fluorescence is generally considered the main contribution of Edmond Becquerel. It had the further impact of leading later to the discovery of radioactivity by his son Henri, as Henri continued th ese studies, including among the substances examined salts of uranium. 

FIGURE 8.11 An energy level diagram depicting the phenomenon of fluorescence in a molecule or complex ion. 

The phenomenon of fluorescence has been synonymous with ultraviolet (UV) and visible spectroscopy rather than near-infrared (near-IR) spectroscopy from the beginning of the subject. This fact is evidenced in definitive texts which also provide useful background information for this volume (see, e.g., Refs. 1-6). Consequently, our understanding of the many molecular phenomena which can be studied with fluorescence techniques, e.g., excimer formation, energy transfer, diffusion, and rotation, is based on measurements made in the UV/visible. Historically, this emphasis was undoubtedly due to the spectral response of the eye and the availability of suitable sources and detectors for the UV/visible in contrast to the lack of equivalent instrumentation for the IR. Nevertheless, there are a few notable exceptions to the prevalence of UV/visible techniques in fluorescence such as the near-IR study of chlorophyll(7) and singlet oxygen,<8) which have been ongoing for some years. 

By analogy, this phenomenon can be compared to that encountered when a hammer hits a rubber block vs. an anvil. With the rubber block, energy will be dissipated in the mass (relaxation) and sound will not be emitted. However, on the anvil, part of the mechanical energy of the hammer will be retransmitted towards the outside (as a loud noise) and this can be compared to the phenomenon of fluorescence of non-rigid vs. rigid molecules. 

The development of photodetectors enabled the human eye to be replaced by a much more sensitive detector of light intensity. The evolution of modem colorimeters and of spectrophotometers capable of operation in both the ultraviolet and visible regions of the spectrum has been discussed.217,218 The phenomenon of fluorescence was first employed for quantitative analysis in the 1930s, when the first filter fluorimeters were constructed. An article has outlined the development of fluorescence analysis up to 1980.219 Lasers have now been employed long enough in analytical chemistry for a historical account to be given.220... 

In the past decade a number of physical techniques have been used to evaluate the unique barrier properties of mammalian skin [1]. This chapter deals with the use of another physical technique, fluorescence spectroscopy, to study the barrier properties of the human stratum corneum (SC), specifically with respect to the transport of ions and water. The SC is the outermost layer of the human epidermis and consists of keratinized epithelial cells (comeo-cytes), physically isolated from one another by extracellular lipids arranged in multiple lamellae [2]. Due to a high diffusive resistance, this extracellular SC lipid matrix is believed to form the major barrier to the transport of ions and water through the human skin [3-5]. The objective of the fluorescence studies described here is to understand how such extraordinary barrier properties are achieved. First the phenomenon of fluorescence is described, followed by an evaluation of the use of anthroyloxy fatty acid fluorescent probes to study the physical properties of solvents and phospholipid membranes. Finally, the technique is applied to the SC to study its diffusional barrier to iodide ions and water. 

In order to appreciate the different methods of data analysis, it is helpful to have a clear idea of the mathematics of the physical phenomenon of fluorescence [9]. On a Jablonski diagram is shown in Figure 1, Sg represents the energy level... 

Fig. 15-1). The primary radiation causes the sample to emit secondary fluorescent radiation, which is then analyzed in a spectrometer. This method, often called fluorescence analysis, is very widely used in industry for chemical analysis. The phenomenon of fluorescence, which is just a nuisance in diffraction experiments, is here made to serve a useful purpose. 

The Raman effect is weak, and its application was severely limited until an extremely strong exciting source, the laser, became available. The principal limitations of Raman spectroscopy now arise from the simultaneous phenomenon of fluorescence, which is a strong interfering signal in the spectrum of many commercial materials. 

The phenomenon of fluorescence can provide information about the physical properties of proteins and other macromolecules. The information content results from the sensitivity of the spectral properties to the average and dynamic properties of the environment surrounding the fluorescent residues. In general, more detailed information is obtainable from time-resolved data than from steady-state measurements. However, the steady-state measurements are considerably easier to perform. At present, the ability to recover time-resolved spectral data is rapidly improving, primarily because of advances in instrument design. The newer instruments may possess resolution adequate to correlate experimental data with the structural or dynamic properties of macromolecules. 

The use of fluorescence microscopy to detect the spin transition in an Fe(II) complex over this range has been proposed recently. The phenomenon of fluorescence is one not generally associated with most Fe(II) SCO complexes." In the present instance, fluorescence originated not from... 

The phenomenon of fluorescence displays a number of general characteristics. Exceptions are known, but these... 

Fluorescence techniques are being used increasingly in wide range of research fields including biochemical, medical and chemical research, due primarily to the inherent sensitivity of the technique and the favorable time scale of the phenomenon of fluorescence (4). Some fluorophores have dramatically different emission characteristics for their protonated and unprotonated forms. Using this phenomenon, the photo-acid concentrations generated within chemically amplified photoresist systems can be monitored (5-7). 

Pigments, which exhibit the phenomenon of fluorescence in the visible region of the spectrum. Few commercial mineral pigments are fluorescent most of the fluorescent pigments are made from fluorescent... 

Raman spectroscopy has suffered for a long time from the phenomenon of fluorescence which interferes with the observed bands. The advent of FT-Raman spectroscopy will hopefully overcome this drawback so that this method will become a successfully cooperating partner to the infrared technique. 

It has often been reported that prolonged exposure of a specimen to a laser beam will produce a Raman spectrum with enhanced signal to noise (the little-understood physical phenomenon of fluorescence burnout which is illustrated in Figure 1) by this means, good quality Raman spectra can be recorded in the presence of fluorescent material. However, the removal of the fluorescence emission is only temporary and the method is not always appropriate. Some biomaterials, such as skin and wool, exhibit strong... 

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