![]() ![]() This reduces the impact of stray light and the incident light wavelength. Even though fluorescence is emitted in all directions from the fluorophores within the sample, fluorescent emissions are detected normal to the incident beam path. Nearly monochromatic light can be produced using a monochromator, where a broad spectrum lamp such as halogen lamp is used ( Figure 2), and the output is passed through a slit onto a diffraction grating, as shown in Figure 3. The sample is typically in solution in a cuvette, and it is excited by near monochromatic light or by monochromatic light from a laser. In fluorescence spectroscopy, also known as fluorometry or spectrofluorometry, fluorescence emissions from a sample are elicited using a range of wavelengths, and the emissions measured. Likewise, when excited by visible light wavelengths, porphyrins emit light in the near-infrared range. For example, when a molecule absorbs short wavelength ultraviolet (UVA) light in the region of 315–400 nm, the emissions may be in the visible spectrum, such as visible red, in the case of porphyrins. This relationship is known as Stokes law and is named after Sir George Stokes, who published the first major paper on fluorescence. The light that is emitted by fluorescence is readily distinguishable from the excitation light because it has a longer wavelength. Molecular fluorescence emissions persist only as long as the incoming stimulating radiation is continued, unlike phosphorescence, where light is emitted as a persisting ‘afterglow’ long after the incoming exciting light is no longer present. Light emission occurs within one microsecond of light exposure. The excited molecule loses energy partly through internal conversion without photon emission, and then it spontaneously releases a lower energy photon as it returns back to the singlet ground state. Based on Ref.Īs the molecule absorbs energy, it transitions from the lower ground singlet state (S 0) to a vibrational level of an excited singlet state S n (n = 1,2,…). Jablonski energy diagram showing fluorescence and phosphorescence processes. This is termed ‘primary fluorescence’ or ‘autofluorescence’. Many naturally occurring substances fluoresce, including some minerals, fungi, bacteria, keratin, collagens and other components of body tissues. Fluorescence and phosphorescence phenomena are illustrated in the Jablonski energy diagram shown in Figure 1. Fluorescence occurs if the transition is between states of the same electron spin and phosphorescence if the transition occurs between states of different spins. When light is absorbed, the fluorophore becomes electronically excited, but the lifetime in the excited state is very short, and there is a rapid decay to a lower energy level. This emission of light occurs as fluorophores get de-excited from a higher energy level to a lower energy level. In fluorescence, the absorption of light of a particular wavelength results in the emission of light of a longer wavelength. Luminescence is a general term for the emission of radiation, which incorporates both fluorescence (a short-lived process) and phosphorescence (a long-lived process), as well as other phenomena such as bioluminescence in living organisms in which chemical reactions generate light. The approaches described have broad applications to clinical and industrial situations where non-invasive detection of microbial biofilms is important. Fluorescence can be used to help discriminate these from healthy tissues. This chapter provides an overview of fluorescence spectroscopic methods for detection and analysis of biofilms and their derivatives such as deposits of dental calculus and how current technology can be extended using photon-counting detectors. Fluorescence is a versatile and powerful diagnostic approach for detection of bacterial biofilms, particularly in dentistry. For bacterial biofilms, bacterial metabolites such as porphyrins are important molecules for diagnostic purposes, since they fluoresce in the red and infrared regions of the spectrum. Efficient detection of biofilms is important for the clinical management of diseases they cause and for providing an endpoint to clinical treatments. Microbial biofilms are complex multi-layered communities of bacteria and fungi which cause a range of oral and other diseases. ![]()
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