Microbial cell autofluorescence as a method for measuring the intracellular content of B2 and B6 vitamins.

Vitamins are important organic compound required for the proper functioning of cells and organisms. Vitamins of special industrial and pharmaceutical interests include riboflavin (vitamin B2) and pyridoxine (vitamin B6). Commercial production of those biological compounds has increasingly relied on microorganisms and requires simple methods for detecting and estimating their level of synthesis during the biotechnological process. In the case of yeast, methods based on autofluorescence, i.e. natural fluorescence emitted by several cellular compounds, including vitamins, may be useful. Considering that the intensity of emitted light is proportional to the intracellular concentration of riboflavin and pyridoxine, autofluorescence may be a convenient method for their quantification. In this report, we demonstrate a simple, rapid, and sufficiently trustworthy spectrofluorimetric method for determining the content of vitamins B2 and B6 in yeast cells which consists of cells growing, harvesting, washing, and resuspending in a buffer, and then measuring the emitted visible light using specific wavelength of excitation (λex=340 nm and λem=385 nm for pyridoxine; λex=460 nm and λem=535 nm for riboflavin). The limits of detection (LOD) and quantification (LOQ) estimated through measurements of vitamin fluorescence were below 0.005 µg/ml for riboflavin and below 0.05 µg/ml for pyridoxine, respectively. In turn, the smallest credible cell density for measuring autofluorescence was set at 1×108 yeast cells/ml. The relative level of the cell's autofluorescence can be expressed in mass units by applying proper calculation formulas. A comparison of the autofluorescence-based method with the reference HPLC-UV method shows that autofluorescence measurement can be used in the screening analysis of vitamin content (especially riboflavin) in microbial cells.

Fatty Acids and Bilirubin as Intrinsic Autofluorescence Serum Biomarkers of Drug Action in a Rat Model of Liver Ischemia and Reperfusion.

The autofluorescence of specific fatty acids, retinoids, and bilirubin in crude serum can reflect changes in liver functional engagement in maintaining systemic metabolic homeostasis. The role of these fluorophores as intrinsic biomarkers of pharmacological actions has been investigated here in rats administered with obeticholic acid (OCA), a Farnesoid-X Receptor (FXR) agonist, proven to counteract the increase of serum bilirubin in hepatic ischemia/reperfusion (I/R) injury. Fluorescence spectroscopy has been applied to an assay serum collected from rats submitted to liver I/R (60/60 min ± OCA administration). The I/R group showed changes in the amplitude and profiles of emission spectra excited at 310 or 366 nm, indicating remarkable alterations in the retinoid and fluorescing fatty acid balance, with a particular increase in arachidonic acid. The I/R group also showed an increase in bilirubin AF, detected in the excitation spectra recorded at 570 nm. OCA greatly reversed the effects observed in the I/R group, confirmed by the biochemical analysis of bilirubin and fatty acids. These results are consistent with a relationship between OCA anti-inflammatory effects and the acknowledged roles of fatty acids as precursors of signaling agents mediating damaging responses to harmful stimuli, supporting serum autofluorescence analysis as a possible direct, real-time, cost-effective tool for pharmacological investigations.

Autofluorescent Biomolecules in Diptera: From Structure to Metabolism and Behavior.

Light-based phenomena in insects have long attracted researchers' attention. Surface color distribution patterns are commonly used for taxonomical purposes, while optically-active structures from Coleoptera cuticle or Lepidoptera wings have inspired technological applications, such as biosensors and energy accumulation devices. In Diptera, besides optically-based phenomena, biomolecules able to fluoresce can act as markers of bio-metabolic, structural and behavioral features. Resilin or chitinous compounds, with their respective blue or green-to-red autofluorescence (AF), are commonly related to biomechanical and structural properties, helpful to clarify the mechanisms underlying substrate adhesion of ectoparasites' leg appendages, or the antennal abilities in tuning sound detection. Metarhodopsin, a red fluorescing photoproduct of rhodopsin, allows to investigate visual mechanisms, whereas NAD(P)H and flavins, commonly relatable to energy metabolism, favor the investigation of sperm vitality. Lipofuscins are AF biomarkers of aging, as well as pteridines, which, similarly to kynurenines, are also exploited in metabolic investigations. Beside the knowledge available in Drosophila melanogaster, a widely used model to study also human disorder and disease mechanisms, here we review optically-based studies in other dipteran species, including mosquitoes and fruit flies, discussing future perspectives for targeted studies with various practical applications, including pest and vector control.

Recent Advances and the Potential for Clinical Use of Autofluorescence Detection of Extra-Ophthalmic Tissues.

The autofluorescence (AF) characteristics of endogenous fluorophores allow the label-free assessment and visualization of cells and tissues of the human body. While AF imaging (AFI) is well-established in ophthalmology, its clinical applications are steadily expanding to other disciplines. This review summarizes clinical advances of AF techniques published during the past decade. A systematic search of the MEDLINE database and Cochrane Library databases was performed to identify clinical AF studies in extra-ophthalmic tissues. In total, 1097 articles were identified, of which 113 from internal medicine, surgery, oral medicine, and dermatology were reviewed. While comparable technological standards exist in diabetology and cardiology, in all other disciplines, comparability between studies is limited due to the number of differing AF techniques and non-standardized imaging and data analysis. Clear evidence was found for skin AF as a surrogate for blood glucose homeostasis or cardiovascular risk grading. In thyroid surgery, foremost, less experienced surgeons may benefit from the AF-guided intraoperative separation of parathyroid from thyroid tissue. There is a growing interest in AF techniques in clinical disciplines, and promising advances have been made during the past decade. However, further research and development are mandatory to overcome the existing limitations and to maximize the clinical benefits.

The Oxidation-Induced Autofluorescence Hypothesis: Red Edge Excitation and Implications for Metabolic Imaging.

Endogenous autofluorescence of biological tissues is an important source of information for biomedical diagnostics. Despite the molecular complexity of biological tissues, the list of commonly known fluorophores is strictly limited. Still, the question of molecular sources of the red and near-infrared excited autofluorescence remains open. In this work we demonstrated that the oxidation products of organic components (lipids, proteins, amino acids, etc.) can serve as the molecular source of such red and near-infrared excited autofluorescence. Using model solutions and cell systems (human keratinocytes) under oxidative stress induced by UV irradiation we demonstrated that oxidation products can contribute significantly to the autofluorescence signal of biological systems in the entire visible range of the spectrum, even at the emission and excitation wavelengths higher than 650 nm. The obtained results suggest the principal possibility to explain the red fluorescence excitation in a large class of biosystems-aggregates of proteins and peptides, cells and tissues-by the impact of oxidation products, since oxidation products are inevitably presented in the tissue. The observed fluorescence signal with broad excitation originated from oxidation products may also lead to the alteration of metabolic imaging results and has to be taken into account.

Endogenous fluorescence can differentiate the keratoconic cornea.

The purpose of the study was to investigate the endogenous fluorescence of the keratoconic cornea in order to analyze changes in the spectra due to the keratoconic stroma abnormalities. Twenty-two corneal buttons obtained from patients with keratoconus (KC, N = 22) at the time of penetrating keratoplasty were used. As a reference, twelve normal corneas (N = 12): ten from the Eye Bank and two from enucleated eyes due to choroidal melanoma were used. The fluorescence excitation/emission matrices (EEM) in the ranges of 250-400/260-600 nm were recorded. Healthy cornea, keratoconic cornea and sclera showed three main EEM bands, which correspond to the following fluorophores: tryptophan residues in the proteoglycan fraction of corneal/scleral stromas, naturally occurring collagen cross-links and the NAD(P)H fraction present in the metabolically active cells. Relative intensity factors S1, S2 and S3 describing the contribution of each kind of fluorophore to the total fluorescence of the tissue were calculated. Normal and keratoconic corneas show qualitatively similar fluorescence matrices, but the statistically significant differences in the mean values of the S1, S2 and S3 parameters for the KC and normal corneas were observed indicating changes in contribution of different fluorophores to the whole fluorescence of the tissue. Moreover, differences between multidimensional distribution of the relative intensity factors S1, S2 and S3 between these groups were demonstrated (p < 0.001). In conclusions: Differences in the relative intensity factors calculated on a basis of the fluorescence spectra can correspond to the changes found in the KC stroma regarding natural collagen cross-links and the proteoglycan fraction. These parameters well differentiate the KC and normal corneas that could serve as an additional tool for the keratoconus characterization.

Distinguishing Healthy and Carcinoma Cell Cultures Using Fluorescence Spectra Decomposition with a Genetic-Algorithm-Based Code.

In this paper, we analysed the steady state fluorescence spectra of cell suspensions containing healthy and carcinoma fibroblast mouse cells, using a genetic-algorithm-spectra-decomposition software (GASpeD). In contrast to other deconvolution algorithms, such as polynomial or linear unmixing software, GASpeD takes into account light scatter. In cell suspensions, light scatter plays an important role as it depends on the number of cells, their size, shape, and coagulation. The measured fluorescence spectra were normalized, smoothed and deconvoluted into four peaks and background. The wavelengths of intensities' maxima of lipopigments (LR), FAD, and free/bound NAD(P)H (AF/AB) of the deconvoluted spectra matched published data. In deconvoluted spectra at pH = 7, the fluorescence intensities of the AF/AB ratio in healthy cells was always higher in comparison to carcinoma cells. In addition, the AF/AB ratio in healthy and carcinoma cells were influenced differently by changes in pH. In mixtures of healthy and carcinoma cells, AF/AB decreases when more than 13% of carcinoma cells are present. Expensive instrumentation is not required, and the software is user friendly. Due to these attributes, we hope that this study will be a first step in the development of new cancer biosensors and treatments with the use of optical fibers.

Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells.

Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time-resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC-5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue- and red-shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra-cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.

Autofluorescence of yeast Saccharomyces cerevisiae cells caused by glucose metabolism products and its methodological implications.

Autofluorescence is the natural fluorescence emitted by cellular compounds which have light emission properties. The main examples of these compounds, identified as an endogenous fluorophores, include aromatic amino acids, vitamins, coenzymes and electron acceptors. As many of them play a critical role in cell metabolism, changes in their content may provide important information on the physiological status of the cell. Nevertheless, the simultaneous occurrence of different endogenous fluorophores in cells makes it difficult to interpret the autofluorescence signal. Autofluorescence values may also be imposed on values obtained through exogenous fluorescent dyes. This study evaluates the origin and the methodological implications of autofluorescence observed in yeast cells. The results show that the level of autofluorescence may differ between yeast cells, which are a result of different concentrations of endogenous fluorophores, including tryptophan, pyridoxine and riboflavin. The study also shows an important influence of autofluorescence on the results obtained by methods based on external fluorescent dyes.

Autofluorescence Spectroscopy for Monitoring Metabolism in Animal Cells and Tissues.

Excitation of biological substrates with light at a suitable wavelength can give rise to a light emission in the ultraviolet (UV)-visible, near-infrared (IR) spectral range, called autofluorescence (AF). This is a widespread phenomenon, ascribable to the general presence of biomolecules acting as endogenous fluorophores (EFs) in the organisms of the whole life kingdom. In cytochemistry and histochemistry, AF is often an unwanted signal enhancing the background and affecting in particular the detection of low signals or rare positive labeling spots of exogenous markers. Conversely, AF is increasingly considered as a powerful diagnostic tool because of its role as an intrinsic biomarker directly dependent on the nature, amount, and microenvironment of the EFs, in a strict relationship with metabolic processes and structural organization of cells and tissues. As a consequence, AF carries multiple information that can be decrypted by a proper analysis of the overall emission signal, allowing the characterization and monitoring of cell metabolism in situ, in real time and in the absence of perturbation from exogenous markers. In the animal kingdom, AF studies at the cellular level take advantage of the essential presence of NAD(P)H and flavins, primarily acting as coenzymes at multiple steps of common metabolic pathways for energy production, reductive biosynthesis and antioxidant defense. Additional EFs such as vitamin A, porphyrins, lipofuscins, proteins, and neuromediators can be detected in different kinds of cells and bulk tissues, and can be exploited as photophysical biomarkers of specific normal or altered morphofunctional properties, from the retinoid storage in the liver to aging processes, metabolic disorders or cell transformation processes. The AF phenomenon involves all living system, and literature reports numerous investigations and diagnostic applications of AF, taking advantage of continuously developing self-assembled or commercial instrumentation and measuring procedures, making almost impossible to provide their comprehensive description. Therefore a brief summary of the history of AF observations and of the development of measuring systems is provided, along with a description of the most common EFs and their metabolic significance. From our direct experience, examples of AF imaging and microspectrofluorometric procedures performed under a single excitation in the near-UV range for cell and tissue metabolism studies are then reported.