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16 pages, 2506 KiB

Open AccessArticle

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

by Alexey N. Semenov, Boris P. Yakimov, Anna A. Rubekina, Dmitry A. Gorin, Vladimir P. Drachev, Mikhail P. Zarubin, Alexander N. Velikanov, Juergen Lademann, Victor V. Fadeev, Alexander V. Priezzhev, Maxim E. Darvin and Evgeny A. Shirshin

Molecules 2020, 25(8), 1863; https://doi.org/10.3390/molecules25081863 - 17 Apr 2020

Cited by 24 | Viewed by 5426

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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13 pages, 1617 KiB

Open AccessFeature PaperArticle

Spectrofluorometric Analysis of Autofluorescing Components of Crude Serum from a Rat Liver Model of Ischemia and Reperfusion

by Anna C. Croce, Andrea Ferrigno, Clarissa Berardo, Giovanni Bottiroli, Mariapia Vairetti and Laura G. Di Pasqua

Molecules 2020, 25(6), 1327; https://doi.org/10.3390/molecules25061327 - 14 Mar 2020

Cited by 7 | Viewed by 2861

Abstract

Autofluorescence (AF) of crude serum was investigated with reference to the potential of its intrinsic AF biomarkers for the noninvasive diagnosis of liver injury. Spectral parameters of pure compounds representing retinol (vitamin A) and fluorescing free fatty acids were characterized by spectrofluorometry, to [...] Read more.

Autofluorescence (AF) of crude serum was investigated with reference to the potential of its intrinsic AF biomarkers for the noninvasive diagnosis of liver injury. Spectral parameters of pure compounds representing retinol (vitamin A) and fluorescing free fatty acids were characterized by spectrofluorometry, to assess spectral parameters for the subsequent AF analysis of serum, collected from rats undergoing liver ischemia/reperfusion (I/R). Differences in AF spectral profiles detected between control and I/R were due to the increase in the AF components representing fatty acids in I/R serum samples. No significant changes occurred for retinol levels, consistently with the literature reporting that constant retinol levels are commonly observed in the blood, except for malnutrition or chronic severe liver disease. Conversely, fatty acids, in particular arachidonic and linoleic acid and their derivatives, act as modulating agents in inflammation, representing both a protective and damaging response to stress stimuli. The biometabolic and pathophysiological meaning of serum components and the possibility of their direct detection by AF spectrofluorometry open up interesting perspectives for the development of AF serum analysis, as a direct, cost effective, supportive tool to assess liver injury and related systemic metabolic alterations, for applications in experimental biomedicine and foreseen translation to the clinics. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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17 pages, 1372 KiB

Open AccessArticle

Study of Methionine Choline Deficient Diet-Induced Steatosis in Mice Using Endogenous Fluorescence Spectroscopy

by Alma Valor, Eduardo J. Arista Romeu, Galileo Escobedo, Adriana Campos-Espinosa, Ivette Irais Romero-Bello, Javier Moreno-González, Diego A. Fabila Bustos, Suren Stolik, Jose Manuel de la Rosa Vázquez and Carolina Guzmán

Molecules 2019, 24(17), 3150; https://doi.org/10.3390/molecules24173150 - 29 Aug 2019

Cited by 6 | Viewed by 2845

Abstract

Non-alcoholic fatty liver disease is a highly prevalent condition worldwide that increases the risk to develop liver fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, it is imperative to develop novel diagnostic tools that together with liver biopsy help to differentiate mild and advanced degrees [...] Read more.

Non-alcoholic fatty liver disease is a highly prevalent condition worldwide that increases the risk to develop liver fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, it is imperative to develop novel diagnostic tools that together with liver biopsy help to differentiate mild and advanced degrees of steatosis. Ex-vivo liver samples were collected from mice fed a methionine-choline deficient diet for two or eight weeks, and from a control group. The degree of hepatic steatosis was histologically evaluated, and fat content was assessed by Oil-Red O staining. On the other hand, fluorescence spectroscopy was used for the assessment of the steatosis progression. Fluorescence spectra were recorded at excitation wavelengths of 330, 365, 385, 405, and 415 nm by establishing surface contact of the fiber optic probe with the liver specimens. A multi-variate statistical approach based on principal component analysis followed by quadratic discriminant analysis was applied to spectral data to obtain classifiers able to distinguish mild and moderate stages of steatosis at the different excitation wavelengths. Receiver Operating Characteristic (ROC) curves were computed to compare classifier’s performances for each one of the five excitation wavelengths and steatosis stages. Optimal sensitivity and specificity were calculated from the corresponding ROC curves using the Youden index. Intensity in the endogenous fluorescence spectra at the given wavelengths progressively increased according to the time of exposure to diet. The area under the curve of the spectra was able to discriminate control liver samples from those with steatosis and differentiate among the time of exposure to the diet for most of the used excitation wavelengths. High specificities and sensitivities were obtained for every case; however, fluorescence spectra obtained by exciting with 405 nm yielded the best results distinguishing between the mentioned classes with a total classification error of 1.5% and optimal sensitivities and specificities better than 98.6% and 99.3%, respectively. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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12 pages, 6673 KiB

Open AccessArticle

Parathyroid Autofluorescence—How Does It Affect Parathyroid and Thyroid Surgery? A 5 Year Experience

by Roland Ladurner, Maximilian Lerchenberger, Norah Al Arabi, Julia K. S. Gallwas, Herbert Stepp and Klaus K. J. Hallfeldt

Molecules 2019, 24(14), 2560; https://doi.org/10.3390/molecules24142560 - 14 Jul 2019

Cited by 45 | Viewed by 4984

Abstract

Injury to parathyroid glands during thyroid and parathyroid surgery is common and postoperative hypoparathyroidism represents a serious complication. Parathyroid glands possess a unique autofluorescence in the near-infrared spectrum which could be used for their identification and protection at an early stage of the [...] Read more.

Injury to parathyroid glands during thyroid and parathyroid surgery is common and postoperative hypoparathyroidism represents a serious complication. Parathyroid glands possess a unique autofluorescence in the near-infrared spectrum which could be used for their identification and protection at an early stage of the operation. In the present study parathyroid autofluorescence was visualized intraoperatively using a standard Storz laparoscopic near-infrared/indocyanine green (NIR/ICG) imaging system with minor modifications to the xenon light source (filtered to emit 690 nm to 790 nm light, less than 1% in the red and green above 470 nm and no blue light). During exposure to NIR light parathyroid tissue was expected to show autofluorescence at 820 nm, captured in the blue channel of the camera. Over a period of 5 years, we investigated 205 parathyroid glands from 117 patients. 179 (87.3%) glands were correctly identified by their autofluorescence. Surrounding structures such as thyroid, lymph nodes, muscle, or adipose tissue did not reveal substantial autofluorescence. We conclude that parathyroid glands can be identified by their unique autofluorescence at an early stage of the operation. This may help to preserve these fragile structures and their vascularization and lower the rate of postoperative hypocalcemia. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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12 pages, 2008 KiB

Open AccessArticle

Autofluorescence Imaging Reflects the Nuclear Enlargement of Tumor Cells as well as the Cell Proliferation Ability and Aberrant Status of the p53, Ki-67, and p16 Genes in Colon Neoplasms

by Kentaro Moriichi, Mikihiro Fujiya, Yu Kobayashi, Yuki Murakami, Takuya Iwama, Takehito Kunogi, Takahiro Sasaki, Masami Ijiri, Keitaro Takahashi, Kazuyuki Tanaka, Aki Sakatani, Katsuyoshi Ando, Yoshiki Nomura, Nobuhiro Ueno, Shin Kashima, Katsuya Ikuta, Hiroki Tanabe, Yusuke Mizukami, Yusuke Saitoh and Toshikatsu Okumura

Molecules 2019, 24(6), 1106; https://doi.org/10.3390/molecules24061106 - 20 Mar 2019

Cited by 6 | Viewed by 2765

Abstract

Background: Autofluorescence imaging (AFI) is useful for diagnosing colon neoplasms, but what affects the AFI intensity remains unclear. This study investigated the association between AFI and the histological characteristics, aberrant methylation status, and aberrant expression in colon neoplasms. Methods: Fifty-three patients with colorectal [...] Read more.

Background: Autofluorescence imaging (AFI) is useful for diagnosing colon neoplasms, but what affects the AFI intensity remains unclear. This study investigated the association between AFI and the histological characteristics, aberrant methylation status, and aberrant expression in colon neoplasms. Methods: Fifty-three patients with colorectal neoplasms who underwent AFI were enrolled. The AFI intensity (F index) was compared with the pathological findings and gene alterations. The F index was calculated using an image analysis software program. The pathological findings were assessed by the tumor crypt density, cell densities, and N/C ratio. The aberrant methylation of p16, E-cadherin, Apc, Runx3, and hMLH1 genes was determined by a methylation-specific polymerase chain reaction. The aberrant expression of p53 and Ki-67 was evaluated by immunohistochemical staining. Results: An increased N/C ratio, the aberrant expression of p53, Ki-67, and the altered methylation of p16 went together with a lower F index. The other pathological findings and the methylation status showed no association with the F index. Conclusions: AFI reflects the nuclear enlargement of tumor cells, the cell proliferation ability, and the altered status of cell proliferation-related genes, indicating that AFI is a useful and practical method for predicting the dysplastic grade of tumor cells and cell proliferation. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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Review

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20 pages, 5118 KiB

Open AccessFeature PaperReview

Autofluorescence in Plants

by Lloyd Donaldson

Molecules 2020, 25(10), 2393; https://doi.org/10.3390/molecules25102393 - 21 May 2020

Cited by 161 | Viewed by 14733

Abstract

Plants contain abundant autofluorescent molecules that can be used for biochemical, physiological, or imaging studies. The two most studied molecules are chlorophyll (orange/red fluorescence) and lignin (blue/green fluorescence). Chlorophyll fluorescence is used to measure the physiological state of plants using handheld devices that [...] Read more.

Plants contain abundant autofluorescent molecules that can be used for biochemical, physiological, or imaging studies. The two most studied molecules are chlorophyll (orange/red fluorescence) and lignin (blue/green fluorescence). Chlorophyll fluorescence is used to measure the physiological state of plants using handheld devices that can measure photosynthesis, linear electron flux, and CO₂ assimilation by directly scanning leaves, or by using reconnaissance imaging from a drone, an aircraft or a satellite. Lignin fluorescence can be used in imaging studies of wood for phenotyping of genetic variants in order to evaluate reaction wood formation, assess chemical modification of wood, and study fundamental cell wall properties using Förster Resonant Energy Transfer (FRET) and other methods. Many other fluorescent molecules have been characterized both within the protoplast and as components of cell walls. Such molecules have fluorescence emissions across the visible spectrum and can potentially be differentiated by spectral imaging or by evaluating their response to change in pH (ferulates) or chemicals such as Naturstoff reagent (flavonoids). Induced autofluorescence using glutaraldehyde fixation has been used to enable imaging of proteins/organelles in the cell protoplast and to allow fluorescence imaging of fungal mycelium. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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33 pages, 2413 KiB

Open AccessReview

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

by Jonas Wizenty, Teresa Schumann, Donna Theil, Martin Stockmann, Johann Pratschke, Frank Tacke, Felix Aigner and Tilo Wuensch

Molecules 2020, 25(9), 2095; https://doi.org/10.3390/molecules25092095 - 30 Apr 2020

Cited by 13 | Viewed by 4176

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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16 pages, 3396 KiB

Open AccessReview

Routine Management of Microalgae Using Autofluorescence from Chlorophyll

by Toshiyuki Takahashi

Molecules 2019, 24(24), 4441; https://doi.org/10.3390/molecules24244441 - 04 Dec 2019

Cited by 32 | Viewed by 6385

Abstract

From a high-potential biomass perspective, microalgae have recently attracted considerable attention due to their extensive application in many areas. Although studies searching for algal species with extensive application potential are ongoing, technical development for their assessment and maintenance of quality in culture are [...] Read more.

From a high-potential biomass perspective, microalgae have recently attracted considerable attention due to their extensive application in many areas. Although studies searching for algal species with extensive application potential are ongoing, technical development for their assessment and maintenance of quality in culture are also critical and inescapable challenges. Considering the sensitivity of microalgae to environmental changes, management of algal quality is one of the top priorities for industrial applications. Helping substitute for conventional methods such as manual hemocytometry, turbidity, and spectrophotometry, this review presents an image-based, automated cell counter with a fluorescence filter to measure chlorophyll autofluorescence emitted by algae. Capturing chlorophyll-bearing cells selectively, the device accomplished precise qualification of algal numbers. The results for cell density using the device with fluorescence detection were almost identical to those obtained using hemocytometry. The automated functions of the device allow operators to reduce working hours, for not only cell density analysis but simultaneous multiparametric analysis such as cell size and algal status based on chlorophyll integrity. The automated device boldly supports further development of algal application and might contribute to opening up more avenues in the microalgal industry. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging)

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