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Fluorescence Microscopy Techniques
Published in Margarida M. Barroso, Xavier Intes, In Vivo, 2020
Mehmet S. Ozturk, Robert Prevedel
The interest in endogenous fluorophores dates back to the early twentie century due to the observed correlation between autofluorescence signal changes and the progression of malignant diseases (Policard, 1924). Since then, fluorescent proteins became a focus of research for both structural and metabolic imaging. Cellular metabolism and intracellular redox states can be visualized with nicotinamide dinucleotide and flavin adenine dinucleotide (NAD, FAD). They can be found in oxidized or reduced states (e.g. NAD+, NADH, respectively) and relate to energy consumption within cells (Ying, 2008). NADP on the other hand plays a key role in biosynthetic pathways. The FAD/NADH fluorescence ratio can be used to quantify the metabolic activity of cells (redox ratio). A decrease in redox ratio indicates an increase in metabolic activity, which is one of the characteristics of cancer cells (Valeur Bernard and Berberan-Santos, 2013). NADH and NADPH have similar fluorescence excitation and emission spectra, 340 ∓ 30 nm and 460 ∓ 50 nm, respectively. The overlapping spectrum makes it difficult to differentiate the origin of the signals, while their distinct fluorescence lifetime facilitates separation by fluorescence lifetime imaging (Blacker et al., 2014).
Biophotonics
Published in Mohammad E. Khosroshahi, Applications of Biophotonics and Nanobiomaterials in Biomedical Engineering, 2017
There are basically two methods for detection of fluorescence: One approach is to use tissue autofluorescence, i.e., the fluorescence originating from naturally occurring fluorophores called endogenous that are specific to normal or dysplastic tissue. The second approach is to use external agents or fluorophores called exogenous that tend to accumulate preferentially within malignant tissue. All tissue exhibit endogenous fluorescence, which is referred to as autofluorescence when is exposed to a particular exciting wavelength. Some of the significant biological fluorophores among others are: Nicotinamide adenine dinucleotide (NADH), Nicotinamide adenine dinucleotide phosphate (NADPH), Flavin adenine dinucleotide (FAD), Flavin mononucleotide (FMN), amino acids, vitamins, and lipids. Some of these are important indicators of tissue metabolic activity. As metabolism is activated, there is a shift to oxidized forms (NAD+, FAD+, etc.). The lack of these oxidized forms results in a reduction of fluorescence signal intensity. Equally important are the other tissue fluorophores, including the connective tissue proteins, collagen, elastin, and porphorines, i.e., the intermediates of haem synthesis (i.e., is a cofactor consisting of an Fe2+ (ferrous) ion contained at the center of a large heterocyclic organic ring called a porphyrin, made up of four pyrrolic groups joined together by methine bridges). Table 6.1 shows the peak excitation and corresponding peak emission wavelengths for some fluorophores in tissue (Bottiroli et al. 1995).
Fluorescence Lifetime Imaging Microscopy of Endogenous Biological Fluorescence
Published in Mary-Ann Mycek, Brian W. Pogue, Handbook of Biomedical Fluorescence, 2003
Endogenous metabolic cofactors, such as reduced pyridine nucleotides (nicotinamide adenine dinucleotide, NADH) and oxidized flavins (flavin adenine dinucleotide, FAD; flavin mononucleotide, FMN), are fluorescent. Because such cofactors are involved in respiratory pathways, monitoring their fluorescence may provide a useful tool for noninvasively assessing metabolic activity. NADH excites in the near-UV (~350 nm) and emits at ~460 nm, whereas flavins excite in the optical region of the electromagnetic spectrum (~450 nm) and emit at ~520 nm. Note that NADH excitation-emission spectra are virtually identical to those of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The abbreviation NAD(P)H is often used to emphasize this indistinguishability. While NADH is used mainly as an electron transporter in cellular respiration, NADPH is used by the cell primarily for its reductive power in biochemical synthesis. Both are present mainly in the mitochondria and cytoplasm. The average fluorescence lifetime of NAD(P)H increases from ~0.4 ns to ~5 ns when protein bound, with a roughly fourfold increase in the quantum yield [2].
Expression and characterization of cholesterol oxidase with high thermal and pH stability from Janthinobacterium agaricidamnosum
Published in Preparative Biochemistry & Biotechnology, 2023
Noriyuki Doukyu, Yuuki Ikehata, Taichi Sasaki
Cholesterol oxidase (COXase, EC 1.1.3.6) is an oxidoreductase containing flavin adenine dinucleotide (FAD) as a redox cofactor. It generally catalyzes the oxidation of the OH group at the C3 position, the isomerization of the double bond between the C5 and C6 positions, and the reduction of oxygen to produce cholest-4-en-3-one (C4E3O) and H2O2.[1,2] However, several COXases oxidize cholesterol to produce 6β-hydroperoxycholest-4-en-3-one (6βHC4E3O) instead of C4E3O.[3,4] COXases have been widely employed to quantify cholesterol content in blood serum for clinical diagnoses.[2] COXases can also be used for pest control due to their pesticidal activity against cotton-eating boll weevil larvae[5] and for the biotransformation of various compounds having 3β-hydroxysteroid structures.[6]Chromobacterium sp. DS-1 COXase alleviates oxysterol cytotoxicity in fibroblasts and thus can have therapeutic applications.[7] Overexpression of Streptomyces gilvosporeus COXase, which serves as a positive regulator of gene expression concerning an antifungal antibiotic, enhances the production of the antibiotic natamycin.[8]
Polarised fluorescence in FAD excited at 355 and 450 nm in water–propylene glycol solutions
Published in Molecular Physics, 2022
D. M. Beltukova, M. K. Danilova, I. A. Gradusov, V. P. Belik, I. V. Semenova, O. S. Vasyutinskii
Optical properties of FAD have been the subject of intensive studies for decades. In his pioneering paper, more than 70 years ago Weber [7] observed the remarkably low fluorescence of FAD with respect to flavin mononucleotide (FMN) and riboflavin (RF) and associated this effect with the formation of a long-living non-fluorescing complex between the Iso and Ad moieties. Most of the authors concluded that FAD in aqueous solution can exist in two conformations: a ‘stack' conformation, where the Iso and Ad moieties are in close proximity with each other that provides electron transfer reactions resulting in fast (picosecond) radiationless deactivation of excited states, and an ‘open’ conformation, where the two moieties are separated from each other [8–11]. The stack conformation is stabilised by a interaction between the Iso and Ad aromatic rings. Only the unfolded conformation can fluoresce, while the stack conformation cannot. In aqueous solution, FAD is considered to predominantly exist in the stack conformation, while an addition of less polar solvents prevents the interaction and produces the open conformation resulting in the increase of the fluorescence quantum yield [11–15]. Molecular dynamic simulations performed for FAD in free and enzyme-bound states demonstrated that FAD may adopt intermediate conformations rather than just the ‘stack’ or ‘open’ ones [16]. The existence of intermediate partially ‘stack’ conformations of FAD was also revealed by Li et al. using aerolysin nanopores [17].
Functional sustainability of nutrient accumulation by periphytic biofilm under temperature fluctuations
Published in Environmental Technology, 2021
Rui Sun, Ying Xu, Yonghong Wu, Jun Tang, Sofia Esquivel-Elizondo, Philip G. Kerr, Philip L. Staddon, Junzhuo Liu
Dehydrogenase is a kind of oxidoreductases and it oxidizes a substrate by reducing an electron acceptor, usually nicotinamide adenine dinucleotide (NAD+)/ nicotinamide adenine dinucleotide phosphate (NADP+), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), and dehydrogenase activity directly correlates with the organic matter degradation capacity of the periphytic biofilm [34,35]. We determined the dehydrogenase activity of the periphytic biofilms in the different temperature phases with results revealing a similar pattern to the utilization of different substrates with temperature variation (Figure 1(d)). Although the temperature increase stimulated the periphytic biofilm carbon metabolic and dehydrogenase activities, the carbon metabolic activity and dehydrogenase activity of the periphytic biofilm reverted to the original level when the temperature fell back to the initial range (17°C).